Methods for multiplex pcr

ABSTRACT

Methods for performing multiplex PCR-based enrichment of a target substrate are provided. Systems and methods for generating a sequencing library are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.17/238,016, filed Apr. 22, 2021, the entire contents of which is herebyincorporated herein by reference. U.S. application Ser. No. 17/238,016is a continuation of U.S. application Ser. No. 16/389,243, filed Apr.19, 2019, now U.S. Pat. No. 11,162,135, the entire contents of which ishereby incorporated herein by reference. U.S. application Ser. No.16/389,243, is a divisional of U.S. application Ser. No. 15/252,397filed Aug. 31, 2016, which is a continuation of U.S. application Ser.No. 15/223,792 filed on Jul. 29, 2016, which is a continuationapplication of International Application No. PCT/US15/13994, filed Jan.30, 2015, which claims priority benefits under 35 U.S.C. § 119 (e) ofProvisional U.S. Patent Application No. 61/934,515, filed Jan. 31, 2014,Provisional U.S. Patent Application No. 62/078,309, filed Nov. 11, 2014,and Provisional U.S. Patent Application No. 62/078,313, filed Nov. 11,2014, the disclosure of each of which is incorporated herein byreference in its entirety.

INCORPORATION BY REFERENCE OF MATERIALS SUBMITTED ELECTRONICALLY

This application contains, as a separate part of disclosure, a SequenceListing in computer readable form (Filename: 47999A_Seqlisting.txt;created Jan. 29, 2015; 47,035 bytes), which is incorporated herein byreference in its entirety.

INTRODUCTION

All commercially available next-generation sequencing (NGS) technologiesrequire library preparation, whereby a pair of specific adaptersequences are ligated to the ends of DNA fragments in order to enablesequencing by the instrument. Most NGS adapters comprise threefunctional domains: (1) unique PCR primer annealing sequences forlibrary and clonal amplification, (2) unique sequencing primer annealingsequences and (3) unique sample indexing sequences. Currently, mostplatforms utilize clonal amplification to make hundreds of copies ofeach individual DNA library molecule. This is achieved by bridgeamplification or emulsion PCR for the purpose of amplifying the signalgenerated for the particular mode of sequence detection for each librarymolecule (e.g., fluorescence or pH). For sequencing by synthesis,annealing domains for sequencing primers are juxtaposed to theadapter-insert junctions; to enable paired-end sequencing, each adapterpossesses a unique sequence for primer annealing. Sample index sequencesare comprised of short unique sequences, typically 6-8 bases, that whensequenced, identify the sample source of a particular sequence read,enabling samples to be multiplexed or co-sequenced. There are existingand emerging single molecule sequencing technologies that do not rely onclonal amplification for signal detection but still require theattachment of adapter sequences to their termini for other purposes,such as adding a terminal hairpin-loop to DNA duplexes to enablesequencing of both strands as a single molecule or introducing a leadersequence for nanopore entry.

Targeted next generation sequencing is encompassed by two leadingtechnologies: amplicon sequencing and hybridization-capture enrichmentof targets from whole genome libraries. Amplicon sequencing is themethod of choice for rapid turnaround time given the reduced number ofsteps, for when panels of target loci significantly smaller than wholeexomes are desired, and for significant overall cost savings in bothpreparative reagents and sequencing depth. Amplicon sequencing isrepresented by a variety of available techniques. Examples of theseinclude 1. Multiplex PCR using degradable target-specific primers toeliminate primer dimers, followed by polishing and NGS adapter ligation,where overlapping targets are divided into separate tubes (Ion TorrentAmpliSeq); 2. Multiplex extension-ligation reactions that incorporateNGS adapters at the termini of each target-specific oligonucleotidepair, followed by NGS adapter mediated PCR amplification, which avoidsmultiplex PCR altogether; however, ligation-mediated PCR requires higherinput DNA quantity (Illumina TSCA); 3. Multiplex PCR on microfluidiccells that separate primer pairs to avoid primer dimer formation andenable overlapping target loci; separate reactions require higher inputDNA quantity (Fluidigm access array); 4. Multiplex PCR by digitaldroplet PCR that also separates primer pairs to avoid primer dimerformation and enable overlapping target loci; also has higher input DNAquantity requirement (Raindance). Each technology is designed toeliminate primer dimers or avoid their formation during the multiplexedamplification process, as to avoid having these artifacts dominate theresulting NGS amplicon library. Drawbacks to existing methods are: A.the high cost of microfluidic or digital droplet instrumentation andconsumables, B. higher input quantity requirements; and C. the necessityto separate multiplex reactions where overlapping or contiguous coverageis desired, thus further increasing input quantity requirements. Analternative to these options when contiguous coverage is desired is toperform long-range PCR. However, long range PCR is difficult tomultiplex and the subsequent fragmentation required for most sequencingplatforms followed by separate NGS library preparation is both timeconsuming and more costly. What is needed in the art is a simple methodof amplicon generation that enables low inputs of approximately 10nanograms (ng) DNA, does not require instrumentation other than athermocycler, and is independent of whether targets are separate hotspotloci or whether targets are overlapping regions of the genome whencontiguous coverage is required. The compositions and methods disclosedherein provides a solution to this need.

Typically, preparation of an NGS DNA library involves 5 steps: (1) DNAfragmentation, (2) polishing, (3) adapter ligation, (4) size selection,and (5) library amplification (See FIGS. 1 and 2).

(1) Fragmentation: Fragmentation of DNA can be achieved by enzymaticdigestion or physical methods such as sonication, nebulization orhydrodynamic shearing. Each fragmentation method has its advantages andlimitations. Enzymatic digestion produces DNA ends that can beefficiently polished and ligated to adapter sequences. However, it isdifficult to control the enzymatic reaction and produce fragments ofpredictable length. In addition, enzymatic fragmentation is frequentlybase-specific thus introducing representation bias into the sequenceanalysis. Physical methods to fragment DNA are more random and DNA sizedistribution can be more easily controlled, but DNA ends produced byphysical fragmentation are damaged and the conventional polishingreaction is insufficient to generate ample ligation-compatible ends.

(2) Polishing: Typical polishing mixtures contain T4 DNA polymerase andT4 polynucleotide kinase (PNK). The 5′-3′ polymerase and the 3′-5′exonuclease activities of T4 DNA polymerase excise 3′ overhangs andfill-in 3′ recessed ends, which results in excision of damaged 3′ basesas well as polishing (creation of blunt) DNA ends. The T4 polynucleotidekinase in the polishing mix adds a phosphate to the 5′ ends of DNAfragments that can be lacking such, thus making them ligation-compatibleto NGS adapters.

What has remained unknown in the art is that a significant number of 5′ends produced by physical fragmentation are damaged in an unidentifiedmanner and do not get phosphorylated by PNK. There is no enzyme in aconventional polishing mix that can trim a damaged 5′ terminal base. Asa result, a substantial fraction of DNA fragments in the preparation donot get converted into NGS library molecules because they remainligation incompatible at their 5′ termini to NGS adapters. Although itis known in the art that adapter ligation is inefficient, ligation istypically performed on both strands simultaneously so it has remainedunknown which strand is limiting. We separated the reactions intostrand-specific ligation to test the efficiency of each, respectively.Through this analysis, we were able to pinpoint the rate limiting stepin the overall process to the 5′ termini which, for a significantfraction of the DNA fragments, are poor substrates for PNK and as aresult, adapter ligation.

(3) Adapter Ligation: Another factor that contributes to low NGS libraryyield apart from a lack of 5′ phosphate groups is the ligation reactionitself. Prior to ligation, adenylation of repaired DNA using a DNApolymerase which lacks 3′-5′ exonuclease activity is often performed inorder to minimize chimera formation and adapter-adapter (dimer) ligationproducts. In these methods, single 3′ A-overhang DNA fragments areligated to single 5′ T-overhang adapters, whereas A-overhang fragmentsand T-overhang adapters have incompatible cohesive ends forself-ligation. However, the adenylation reaction is incomplete andgenerates non-specific side products, further reducing the number ofavailable molecules for ligation which reduces library yield. A moreefficient, alternative approach to minimize concatamer formation ispresented herein.

(4) Size Selection: The size selection process also impacts libraryyield. During size selection, fragments of undesired size are eliminatedfrom the library using gel or bead-based selection in order to optimizethe library insert size for the desired sequencing read length. Thismaximizes sequence data output by minimizing overlap of paired endsequencing that occurs from short DNA library inserts. In the case ofsamples with extremely limited input quantities, this step can beskipped, and in exchange for a higher degree of paired-end overlap, morerare fragments are sequenced.

(5) Amplification: The problem of low library yield results in thenecessity to amplify libraries by PCR prior to NGS analysis, which leadsto loss of library complexity and introduction of base composition bias.The only current solution to avoid this problem is higher quantities ofinput DNA for library prep, but up to 20% of clinical samples submittedfor NGS analysis have insufficient DNA quantity, so instead, additionalPCR cycles are applied to overcome the insufficient DNA input. Thisresults in reduced sequence data from the presence of an unacceptablepercentage of PCR duplicates.

SUMMARY OF THE INVENTION

To address some of the existing problems described above which cause lowyields for NGS library construction, an enhanced adapter ligation methodis provided. This novel method overcomes the necessity to add aphosphate group to the 5′ ends of DNA fragments (which is required forconventional adapter ligation; see FIGS. 1 and 2). Instead, the 5′terminal bases that are damaged as a result of physical fragmentation ofthe DNA, are removed. By removal of the damaged base, a ligationcompatible base with a 5′ phosphate is exposed and adapter ligationefficiency is restored, leading to a significant increase in libraryyield and the ability to construct libraries from reduced input DNAquantities. In addition, an alternative to adenylation/TA ligation forthe prevention of chimeric library inserts (concatamer formation duringligation) and formation of adapter dimer ligation products isintroduced, which also contributes to higher library preparation yields.In any embodiment of a method described herein, the processing comprisesconverting the 5′ and/or 3′ terminus of a substrate molecule to one thatis ligation-compatible.

This method, in its exemplary form, is comprised of four separateincubations (see FIGS. 3, 4 and 5) to generate a processed substratemolecule. In the first incubation, double-stranded fragmented DNA iscombined with a phosphatase enzyme and under appropriate reactionconditions, the enzyme removes phosphate groups from the termini of theDNA fragments. This prevents chimeric library inserts from beinggenerated by preventing DNA fragment concatamer formation in thesubsequent ligation reactions.

In the second incubation, the de-phosphorylated DNA fragments arecombined with a polymerase or a cocktail of polymerases that possess3′-5′ exonuclease activity. Under appropriate reaction conditions and inthe presence of dNTPs, damaged 3′ bases are trimmed and polishing of thedouble-stranded DNA fragments is achieved by excision of 3′ overhangsand filling in of 3′ recessed ends that were generated during physicalfragmentation. At the completion of this step, the DNA fragments possessblunt ends with ligation compatible 3′ termini and 5′ termini which lackphosphate groups, therefore rendering the DNA fragments incapable ofself-ligation.

In the third incubation, the blunt ended, double-stranded DNA fragmentsare combined with a DNA ligase and a first double-stranded blunt endedNGS adapter (3′ adapter) that comprises a 5′ phosphate and which iscapable of ligating to the 3′ ends of the DNA fragments (see FIG. 3).The special feature of this 3′ adapter is that the adapter DNA strandthat would typically simultaneously ligate to the 5′ end of the DNAfragments has a 3′ end modification that prevents ligation, andtherefore a nick remains at the junction of the 5′ terminus of each DNAfragment and the 3′ end of the 3′ adapter following the ligationreaction even in the presence of the 5′ phosphate. The same 3′modification that prevents ligation to the 5′ termini of the DNAfragments also prevents adapter-adapter ligation products from forming,albeit they would be comprised of a single adapter sequence which wouldnot be a functional adapter dimer (functional dimers are comprised ofboth adapters). The product of this step is double-stranded DNAfragments with a single NGS adapter ligated to only one strand on both3′ termini.

In the fourth incubation, the strand of the 3′ adapter that remainsunligated to the DNA fragments (due to the 3′ modification) is alsodisplaceable or degradable due to the incorporation of degradable basesduring oligo synthesis. In the presence of an optional, appropriateenzyme during the fourth incubation, the 3′ adapter strand is degradedor is displaced by a new single-stranded adapter comprising the secondNGS adapter sequence that is also present in the reaction (5′ adapter,see FIG. 3), and through a complementary sequence to the 3′ adapter atthe junction of the adapter-insert, the single-stranded 5′ adapteranneals to the complementary portion of the 3′ adapter that is ligatedto the 3′ ends of the double-stranded DNA fragments, resulting in therestoration of a nick or gap. Additionally in the reaction is a DNApolymerase that possesses 5′-3′ exonuclease activity, and in thepresence of dNTPs, a ligase and the appropriate reaction conditions,nick translation is initiated at the nick or gap residing at thejunction of the 5′ adapter and the 5′ termini of the DNA fragments. Nicktranslation results in replacement of the damaged 5′ terminal base (andan additional one or more bases internal to the 5′ terminus) and exposesa ligation-compatible 5′ terminal phosphate group. Subsequently,efficient ligation of the 5′ adapter to the DNA substrate moleculeoccurs when ligase seals the nick that is translated one or more bases(see FIG. 4). At the completion of this novel adapter ligation process,both ends of each double-stranded DNA fragment are flanked by twodifferent, single-stranded NGS adapters that share a short complementaryadapter sequence at the adapter-insert junction.

Alternatively, removal of the 5′ terminal base and 5′ adapter ligationcan be achieved without polymerization, by annealing the single-stranded5′ adapter with one or more additional random bases at its 3′ terminuswhich overlaps with the damaged 5′ base(s) of the substrate molecule,and in the absence of dNTPs, cleavage of the displaced base at the 5′terminus of DNA substrate molecules by a 5′ flap-specific nucleaseoccurs following displacement, which results in efficient ligation ofthe second NGS adapter to the exposed 5′ phosphate on the termini of thecleaved DNA substrate molecules (see FIG. 5).

In another alternative, 5′ terminal base removal and 5′ adapter ligationcan be achieved by a single dideoxy base extension from the degradableor displaceable strand of the 3′ adapter that is followed by cleavage ofthe 5′ terminal base of the DNA fragments by the 5′ flap endonucleaseactivity of the polymerase. The strand is then degraded or displaced bythe 5′ adapter, and in the presence of a ligase, the 5′ adapterefficiently ligates to the exposed 5′ phosphate on the DNA fragments.Alternative embodiments of this step and preceding steps are presentedbelow.

Accordingly, in one aspect the disclosure provides a method of producinga processed substrate molecule, the method comprising (i) ligating afirst polynucleotide to a 3′ terminus of a substrate molecule that is atleast partially double stranded; (ii) annealing a second polynucleotideto the first polynucleotide under conditions that promote the annealing;(iii) excising at least one nucleotide from the 5′ terminus of thesubstrate molecule; and then (iv) ligating the second polynucleotide tothe 5′ terminus of the double stranded substrate molecule to produce theprocessed substrate molecule. In one embodiment, the method furthercomprises the step, prior to step (i), of contacting the substratemolecule with a phosphatase enzyme. In another embodiment, the methodfurther comprises the step of making the substrate molecule blunt-endedby contacting the substrate molecule with a polymerase enzyme possessing3′-5′ exonuclease activity. In yet another embodiment, the methodfurther comprises the step of contacting the substrate molecule with atemplate-independent polymerase to adenylate the 3′ end of the substratemolecule.

In any of the methods disclosed herein, it is contemplated that thesubstrate molecule is naturally occurring or the substrate molecule issynthetic. In one embodiment, the substrate molecule is naturallyoccurring. In another embodiment, the substrate molecule is genomic DNA,and in further embodiments the genomic DNA is eukaryotic or prokaryotic.In embodiments in which the substrate molecule is genomic DNA, thedisclosure contemplates that the genomic DNA is fragmented in vitro orin vivo. In some embodiments, the in vitro fragmenting is performed by aprocess selected from the group consisting of shearing, cleaving with anendonuclease, sonication, heating, irradiation using an alpha, beta, orgamma source, chemical cleavage in the presence of metal ions, radicalcleavage, and a combination thereof. In some embodiments, the in vivofragmenting occurs by a process selected from the group consisting ofapoptosis, radiation, and exposure to asbestos.

The disclosure also contemplates embodiments in which the substratemolecule is synthetic and is selected from the group consisting of cDNA,DNA produced by whole genome amplification, primer extension productscomprising at least one double-stranded terminus, and a PCR amplicon.

In any of the aspects or embodiments of the disclosure, it iscontemplated that the first polynucleotide is at least partially doublestranded and comprises oligonucleotide 1 and oligonucleotide 2. In someembodiments, the second polynucleotide anneals to oligonucleotide 1, andin further embodiments, the annealing results in a nick, a gap, or anoverlapping base between the second polynucleotide and the substratemolecule. In some embodiments, the annealing results in dehybridizationof oligonucleotide 1 and oligonucleotide 2.

The second polynucleotide, in various embodiments, is contacted with apolymerase, resulting in degradation of oligonucleotide 2.

Also contemplated by the disclosure are embodiments whereinoligonucleotide 2 comprises a base that is susceptible to degradation,and the disclosure also provides embodiments wherein oligonucleotide 2comprises a blocking group at its 3′ end that prevents ligation. In someembodiments, the second polynucleotide comprises a modified base.

In further embodiments, a method of the disclosure further comprises (i)ligating a third polynucleotide to a 3′ terminus of an additionalsubstrate molecule that is at least partially double stranded; (ii)annealing a fourth polynucleotide to the third polynucleotide underconditions that promote the annealing; (iii) excising at least onenucleotide from the 5′ terminus of the additional substrate molecule;and then (iv) ligating the fourth polynucleotide to the 5′ terminus ofthe double stranded additional substrate molecule to produce a processedadditional substrate molecule. In some embodiments, the firstpolynucleotide and the third polynucleotide are the same. In someembodiments, the second polynucleotide and the fourth polynucleotide arethe same.

The method of targeted amplicon NGS library construction comprises twoseparate steps: multiplex PCR target enrichment followed by an NGSadapter ligation step (see FIG. 39). Two separate workflow options arepossible: a two-step PCR followed by adapter ligation or a one-step PCRfollowed by ligation.

In the multiplex PCR step using either method, pairs of target-specificprimers are designed to desired target loci and comprise a universaltruncated NGS adapter sequence at their 5′ termini (see FIGS. 40 and 41,Table 2). The first PCR cycles have elongated cycling times to allow thehigh complexity of primer pairs, each of which is at a lowconcentration, to create universal NGS adapter tagged amplicons fromtheir target sequences. These primers optionally possess uniquedegenerate sequence tags to identify individual amplicons (UI=uniqueidentifier), where each UI is located between the universal NGS adaptersequence at the 5′ terminus and the target-specific portion at the 3′terminus of each primer (and represented as a stretch of NNNN bases,FIGS. 40, 41). If UI sequences are used, the elongated multiplex PCRcycles are limited to 2 in order to avoid incorporation of additional UIsequences into copies of previously generated amplicons; if UI sequencesare not used, the elongated multiplex PCR cycles can be performed formore than 2 cycles. The more limited the target-specific cycle numberperformed, the fewer primer dimer products that accumulate, so theminimum number of multiplexed cycles feasible for the input samplequantity should be performed. Following the multiplex cycles (2 ormore), PCR is continued with shorter elongation times for a second phaseof amplification using a single, universal primer that corresponds tothe universal truncated NGS adapter flanking each target amplicon. Theuniversal primer is used at a relatively high concentration compared tothe target-specific primers, where the total number of cycles isdetermined by the desired library yield. The concentration oftarget-specific primers are not sufficient to amplify the targets, sothe universal primer which cannot self-interact, takes over theamplification reaction with the absence of additional primer dimerformation. Additionally, the primer dimers that accumulate during thelimited multiplexed cycles will be shorter in length than the desiredamplicons and will be subject to stable secondary structure whichresults in less efficient amplification by the single universal primer.If UI sequences are used, a purification step or exonuclease I digestionof multiplex primers is required prior to addition of the universalprimer, in order to prevent additional UI sequences labeling subsequentcopies of previously generated amplicons. If UI sequences are not used,the universal primer can be added at the beginning of the reaction withthe multiplex primers and will become functional once universal adaptertagged amplicons are generated.

An additional feature of the universal primer is that it optionallycomprises cleavable bases to enable downstream adapter ligation. Withoutlimitation, the cleavable bases can be comprised of deoxyuridine, RNA ordeoxyinosine. Alternatively, the universal primer does not comprisecleavable bases and this sequence is later excised using a 5′exonuclease to enable adapter ligation (see FIG. 42). In addition, bothtarget-specific primers and the universal primer optionally comprisenuclease-resistant modifications at their 3′ termini; these includephosphorothioate linkages, 2′O-Methyl or methylphosphonatemodifications. These enable more specific and efficient priming whenusing a proofreading polymerase that possesses 3′ to 5′ exonucleaseactivity. It also limits 5′ exonuclease digestion if this enzyme is usedto remove the universal adapter sequence from amplicons prior to adapterligation. Following PCR, a purification step is required to remove theunused reagents and polymerase.

For the final step of adapter ligation (see FIG. 42), the portion ofeach amplicon derived from the universal primer is digested due toincorporation of degradable bases into the primer and use ofmodification-specific endonuclease. Alternatively, for primerscontaining nuclease-resistant bases at their 3′ end, the 5′ portion ofeach amplicon can be trimmed by 5′ exonuclease digestion. In this case,exonuclease digestion of the 5′ termini of amplicons will be terminatedat the position of the nuclease-resistant base. The primer digestionreaction creates a single stranded 3′ overhang on both termini of eachamplicon. Also present in the reaction is a full-length, single-strandedadapter B comprising a second NGS adapter sequence, and through acomplementary sequence to the universal adapter at the junction of theadapter-target, the single-stranded second adapter B anneals to thecomplementary portion of the universal adapter that is located at the 3′overhangs of each amplicon, where the adapter annealing results in theformation of a nick or gap. Additionally in the reaction is a DNApolymerase that possesses 5′-3′ exonuclease activity, and in thepresence of dNTPs, a ligase and the appropriate reaction conditions,nick translation is initiated at the nick or gap residing at thejunction of adapter B and the 5′ termini of the amplicons. Nicktranslation results in replacement of one or more bases internal to the5′ terminus and exposes a ligation-compatible 5′ terminal phosphategroup. Subsequently, efficient ligation of adapter B to the DNAsubstrate amplicon occurs when ligase seals the nick that is translatedone or more bases. Alternatively, ligation of adapter B is accomplishedby a displacement-cleavage reaction using a polymerase with flapendonuclease activity and additionally a ligase. In this case, dNTPs arenot required, only a several base overlap between the 3′ terminus ofAdapter B and the 5′ terminus of the universal adapter portion remainingon each amplicon. To complete the adapter ligation process, alinker-mediated ligation is simultaneously performed to complete the1^(st) adapter (A) on the remaining universal adapter sequence at the 3′end of each amplicon. The linker oligonucleotide is complementary to the3′ terminus of the remaining universal adapter on each amplicon andcomplementary to the oligonucleotide comprising the remainder of the 1″adapter. Through its complementarity to both sequences, the linkeroligonucleotide hybridizes to both the 3′ remainder of the 1^(st)adapter and the remaining universal adapter present on each amplicon,enabling ligation to occur. At the completion of this novel adapterligation process, both ends of each amplicon are flanked by twodifferent, single-stranded NGS adapters (A and B) that share a shortcomplementary adapter sequence at the adapter-target junction. A finalpurification step prior to library quantification and sequencing is thenperformed.

An additional feature of the disclosed method is the choice of DNApolymerase used in the multiplexed PCR amplification reaction. The errorrate during amplification can be improved when using high fidelity PfuDNA polymerase, Phusion DNA polymerase, KAPA HiFi DNA polymerase, Q5 DNApolymerase or their derivatives and analogs. Additionally, given thatthe universal primer used in the second phase of the amplificationreaction optionally comprises cleavable bases, a high fidelity DNApolymerase that is tolerant of uracil, RNA or inosine bases is alsodesirable. This includes but is not limited to KAPA HiFi U+ polymerase,Themo Phusion U and Enzymatics VeraSeq ULtra polymerases, all engineeredto tolerate uracil containing substrates. Given the use of high fidelityenzymes that possess 3′ to 5′ exonuclease activity in the amplificationreaction, all target-specific primers as well as the universal primercomprise nuclease resistant linkages at their 3′ termini to increase thefidelity and efficiency of primer extension. This includes but is notlimited to a phosphorothioate linkage or other nuclease resistantmoiety.

Additionally, as previously mentioned, methods for multiplexed PCR fortargeted NGS libraries that are capable of amplifying overlappingtargets for contiguous coverage in a single tube format is desired. Themethod disclosed herein is capable of achieving this effect (FIGS. 43and 44). In the case of two primer pairs that have overlapping targetregions, 4 possible amplicons can be generated: an amplicon specific toeach of the two primer pairs, a maxi-amplicon resulting fromamplification of the two distal primers and a mini-amplicon resultingfrom amplification of the two proximal primers. To avoid having themini-amplicon dominate the multiplexed PCR reaction (short ampliconssuch as this and primer dimers often dominate amplification reactionsdue to their short length and ease of amplification), most methodsseparate overlapping primer pairs into two tubes, which is effective butdoubles the workload and required DNA input quantity. The methoddisclosed herein enables overlapping amplicons to be created in a singletube, because due to the presence of the universal sequence at eachterminus, the short mini-amplicon will be subject to stable secondarystructure which results in less efficient amplification by the singleuniversal primer. Therefore, even if the mini-amplicon is producedduring the initial target-specific PCR cycles, it will not beefficiently amplified. As a result, using methods disclosed herein, onlythe amplicons specific to each primer pair and the maxi-amplicon areproduced from high quality, high molecular weight DNA input. Whencross-linked FFPE DNA or fragmented DNA (particularly circulatingcell-free DNA that is in the 165 bp range) is used, formation of themaxi-amplicon is suppressed since template length or integrity cannotsupport an amplicon of this size, and only the amplicons specific toeach primer pair are produced.

In any of the methods disclosed herein, it is contemplated that thesample DNA input is naturally occurring. In one embodiment, the inputDNA is genomic DNA, either intact high molecular weight DNA orfragmented circulating cell-free DNA, and in further embodiments, thegenomic DNA is eukaryotic, prokaryotic, mitochondrial or viral inorigin. In other embodiments, the input DNA is single-stranded ordouble-stranded or is synthetic and is the result of a prior wholegenome amplification or the result of a random or otherwise primedreverse transcription of RNA.

In further aspects of the disclosure, a composition is providedcomprising a ligase and a first polynucleotide that is at leastpartially double stranded and comprises oligonucleotide 1 andoligonucleotide 2; wherein oligonucleotide 1 comprises a 5′ phosphateand a blocking group at its 3′ terminus; and wherein oligonucleotide 2(i) comprises a base that is susceptible to degradation and/or (ii) canbe displaced by non-denaturing heat condition and further comprises ablocking group at its 3′ end, said blocking group prevents ligation ofthe 3′end but enables ligation of the 5′ end of oligonucleotide 1.

In some embodiments, the 3′ blocking group of oligonucleotide 2 is 3′deoxythymidine, 3′ deoxyadenine, 3′ deoxyguanine, 3′ deoxycytosine or adideoxy nucleotide. In further embodiments, the base that is susceptibleto degradation is deoxyuridine, a ribonucleotide, deoxyinosine, orinosine. The non-denaturing heat condition, in various embodiments, isfrom about 50° C. to about 85° C.

In some embodiments, oligonucleotide 2 comprises a base modificationthat reduces the binding stability of oligonucleotide 2, wherein thebase modification is deoxyinosine, inosine or a universal base.

The disclosure also provides, in some aspects, a composition comprisinga ligation product resulting from incubation of a double strandedsubstrate with a composition of the disclosure; a ligase, a DNApolymerase having nick translation activity, an endonuclease thatrecognizes a base that is susceptible to degradation, and a secondpolynucleotide that is single stranded and comprises a 3′ domain that issufficiently complementary to the 5′ portion of oligonucleotide 1 ofpolynucleotide 2 to anneal under appropriate conditions whenoligonucleotide 2 of polynucleotide 1 is either degraded or displaced.

In some embodiments, the second polynucleotide is of a sufficient lengthto displace oligonucleotide 2 of the first polynucleotide or the secondpolynucleotide comprises a base modification that increases its bindingstability. In further embodiments, the endonuclease is selected from thegroup consisting of UDG plus endonuclease VIII, RNase HI, RNase H2 andEndonuclease V. In still further embodiments, the ligase is E. coli DNAligase or T4 DNA ligase. The base modification that increases itsbinding stability is, in various embodiments, a locked nucleic acid(LNA).

In further aspects of the disclosure, a composition is providedcomprising a ligation product resulting from incubation of a doublestranded substrate with a composition of the disclosure; a ligase; aflap endonuclease; an endonuclease that recognizes a base that issusceptible to degradation; a second polynucleotide comprising a singlestranded oligonucleotide comprising a 3′ domain that is sufficientlycomplementary to the 5′ portion of oligonucleotide 1 of polynucleotide 2to anneal under appropriate conditions when oligonucleotide 2 ofpolynucleotide 1 is either degraded or displaced, wherein the secondpolynucleotide is of a sufficient length to displace oligonucleotide 2of the first polynucleotide or the second polynucleotide comprises abase modification that increases its binding stability, and wherein thesecond polynucleotide further comprises a 3′ terminal degenerate base.

In another aspect, the disclosure provides a method of producing aprocessed substrate molecule, the method comprising: (i) ligating afirst polynucleotide to a 3′ terminus of a substrate molecule that is atleast partially double stranded; (ii) annealing a second polynucleotideto the first polynucleotide under conditions that promote the annealing;(iii) excising at least one nucleotide from the 5′ terminus of thesubstrate molecule; and then (iv) ligating the second polynucleotide tothe 5′ terminus of the double stranded substrate molecule to produce theprocessed substrate molecule. In some embodiments, the method furthercomprises a step, prior to step (i), of contacting the substratemolecule with a phosphatase enzyme.

In some embodiments, the phosphatase enzyme is calf intestinalphosphatase or shrimp phosphatase.

In further embodiments, the method further comprises a step of makingthe substrate molecule blunt-ended by contacting the substrate moleculewith a polymerase enzyme possessing 3′-5′ exonuclease activity.

In some embodiments, the polymerase enzyme is selected from the groupconsisting of T4 DNA ligase, Klenow fragment, T7 polymerase, and acombination thereof. In still further embodiments, the method furthercomprises a step of contacting the substrate molecule with atemplate-independent polymerase to adenylate the 3′ end of the substratemolecule.

In various embodiments, the substrate molecule is naturally occurring orthe substrate molecule is synthetic. Thus, in some embodiments, thesubstrate molecule is naturally occurring. In further embodiments, thesubstrate molecule is genomic DNA. In still further embodiments, thegenomic DNA is eukaryotic or prokaryotic, and in yet additionalembodiments, the genomic DNA is fragmented in vitro or in vivo. In someembodiments, the substrate molecule is circulating cell-free DNA.

In some embodiments, the method further comprises, prior to step (i),adjusting temperature to between about 50° C. to about 85° C. In someembodiments, the temperature is 65° C.

In additional embodiments, the in vitro fragmenting is performed by aprocess selected from the group consisting of shearing, cleaving with anendonuclease, sonication, heating, irradiation using an alpha, beta, orgamma source, chemical cleavage in the presence of metal ions, radicalcleavage, and a combination thereof. In further embodiments, the in vivofragmenting occurs by a process selected from the group consisting ofapoptosis, radiation, and exposure to asbestos.

The substrate molecule, in further embodiments, is synthetic and isselected from the group consisting of cDNA, DNA produced by whole genomeamplification, primer extension products comprising at least onedouble-stranded terminus, and a PCR amplicon.

In some embodiments, the first polynucleotide is at least partiallydouble stranded and comprises oligonucleotide 1 and oligonucleotide 2.In various embodiments, the second polynucleotide anneals tooligonucleotide 1. The annealing, in some embodiments, results in anick, a gap, or an overlapping base between the second polynucleotideand the substrate molecule.

The second polynucleotide, in various embodiments, is contacted with apolymerase, resulting in degradation of oligonucleotide 2.

In some embodiments, oligonucleotide 2 comprises a base that issusceptible to degradation. In further embodiments, the base that issusceptible to degradation is selected from the group consisting ofdeoxyuridine, RNA, deoxyinosine, and inosine. In still furtherembodiments, oligonucleotide 2 comprises a blocking group at its 3′ endthat prevents ligation. the blocking group, in various embodiments, is a3′ deoxynucleotide or a dideoxynucleotide.

In some embodiments, the second polynucleotide comprises a modifiedbase.

In further embodiments, the annealing results in dehybridization ofoligonucleotide 1 and oligonucleotide 2.

In still further embodiments, the method further comprises: (i) ligatinga third polynucleotide to a 3′ terminus of an additional substratemolecule that is at least partially double stranded; (ii) annealing afourth polynucleotide to the third polynucleotide under conditions thatpromote the annealing; (iii) excising at least one nucleotide from the5′ terminus of the additional substrate molecule; and then (iv) ligatingthe fourth polynucleotide to the 5′ terminus of the double strandedadditional substrate molecule to produce a processed additionalsubstrate molecule.

In some embodiments, the first polynucleotide and the thirdpolynucleotide are the same. In further embodiments, the secondpolynucleotide and the fourth polynucleotide are the same.

In further aspects, the disclosure provides a composition comprising auniversal primer and a plurality of target-specific oligonucleotideprimer pairs; wherein each target-specific primer of the plurality ofprimer pairs comprises a target-specific sequence and a 5′ terminalsequence that is not complementary to a target substrate molecule;wherein the universal primer comprises the 5′ terminal sequence and acleavable base or a nuclease resistant modification; wherein eachtarget-specific primer of the plurality of primer pairs and theuniversal primer each comprise a nuclease resistant modification attheir 3′ termini; a high fidelity polymerase that is tolerant of thecleavable base incorporated into the universal primer; wherein thetarget-specific primer pairs and the universal primer anneal to theirtarget substrate molecules at the same temperature; and wherein themolar ratio of target-specific to universal primer is at least about1:100.

In some embodiments, the cleavable base is deoxyuridine, RNA,deoxyinosine, or inosine. In further embodiments, the nuclease resistantmodification is phosphorothioate.

In additional embodiments, at least one target-specific primer furthercomprises a molecular identification tag between the target-specificsequence and the 5′ terminal sequence.

In various embodiments of the disclosure, the molar ratio oftarget-specific to universal primer is at least about 1:200, or at leastabout 1:300, or at least about 1:400, or at least about 1:500, or atleast about 1:1000, or at least about 1:2000, or at least about 1:3000,or at least about 1:5000, or at least about 1:10,000 or greater.

In various embodiments, the composition further comprises a substratemolecule.

In some aspects, a composition is provided comprising a product of apolymerase chain reaction (PCR) generated by a universal primer, whereinthe product comprises at least one cleavable base incorporated via theuniversal primer; an endonuclease that can cleave the cleavable base;(i) at least one nucleotide and a DNA polymerase possessing nicktranslation activity, or (ii) an enzyme possessing flap endonucleaseactivity; a DNA ligase; a 5′ adapter comprising (i) a 3′ sequence thatis complementary to the 5′ portion of the reverse complement of theuniversal primer exposed by endonuclease cleavage of the universalprimer and (ii) a 5′ portion that is not complementary to the reversecomplement of the universal primer; and wherein the 3′ portion of thereverse complement of the universal primer anneals to a partially doublestranded truncated 3′ adapter.

In some embodiments, the endonuclease is selected from the groupconsisting of UDG+Endonuclease VIII, RNase HI, RNase H2, andEndonuclease V. In further embodiments, the DNA ligase is E. coli DNAligase or T4 DNA ligase.

In further aspects, the disclosure provides a composition comprising: aproduct of a polymerase chain reaction (PCR) generated by a universalprimer, wherein the product comprises at least one nuclease resistantmodification incorporated via the universal primer; a 5′ exonucleasethat is not able to digest the PCR product beyond the nuclease resistantmodification; (i) at least one nucleotide and a DNA polymerasepossessing nick translation activity, or (ii) an enzyme possessing flapendonuclease activity; a DNA ligase; 5′ adapter comprising (i) a 3′sequence that is complementary to the 5′ portion of the reversecomplement of the universal primer exposed by endonuclease cleavage ofthe universal primer and (ii) a 5′ portion that is not complementary tothe reverse complement of the universal primer; and wherein the 3′portion of the reverse complement of the universal primer anneals to apartially double stranded truncated 3′ adapter molecule.

In still further aspects, a method of polymerase chain reaction (PCR) isprovided comprising contacting a substrate molecule with: (i) atarget-specific primer pair, where each primer comprises a 5′ sequencethat is not complementary to the substrate molecule and whichincorporates a single universal adapter at the termini of the resultingamplicon; and (ii) a single primer that comprises the single universaladapter sequence and additionally comprises a cleavable base or nucleaseresistant modification, where under appropriate reaction conditionsusing a constant annealing temperature for each cycle of PCR but varyingthe annealing time, in the presence of a high fidelity DNA polymeraseand nucleotides, wherein the molar ratio of each target-specific primer:universal primer is at least about 1:100, target-specific amplicons aregenerated during the first two or more PCR cycles that have annealingtimes of 5 minutes or more, followed by amplification of the resultingamplicons during the remaining PCR cycles which each comprise annealingtimes of 1 minute or less, wherein amplification of the target-specificamplicon by the higher concentration single universal primer isachieved.

In additional aspects, a method of multiplexed PCR is providedcomprising contacting a substrate molecule with (i) a plurality oftarget-specific primer pairs, wherein each primer comprises a 5′sequence that is not complementary to the substrate and whichincorporates a single universal adapter at the termini of the resultingamplicon, and (ii) a single primer that comprises the single universaladapter sequence and additionally comprises a cleavable base or nucleaseresistant modification, where under appropriate reaction conditionsusing a constant annealing temperature for each PCR cycle but varyingthe annealing time, in the presence of a high fidelity DNA polymeraseand nucleotides, wherein the molar ratio of each target-specific primer:universal primer is at least about 1:100, target-specific amplicons aregenerated during the first two or more PCR cycles that have annealingtimes of five minutes or more, followed by amplification of theresulting amplicons during the remaining PCR cycles which each comprisean annealing time of one minute or less, wherein multiplexedamplification of the target-specific amplicons by the higherconcentration single universal primer is achieved.

In some aspects, a method of converting a polymerase chain reaction(PCR) product is provided, comprising a single universal adaptersequence at each terminus into a product comprising asymmetric 5′ and 3′adapters at each terminus, comprising: (a) digesting the 5′ terminus ofthe PCR product where either a cleavable base or nuclease resistantmodification was introduced, followed by (b) annealing and (i)nick-translation ligation or (ii) flap endonuclease cleavage ligation ofa 5′ adapter that is complementary to the 5′ portion of the reversecomplement of the universal adapter that was exposed by the digestion,and (c) wherein a partially double stranded truncated 3′ adapter annealsand ligates to the 3′ portion of the reverse complement of the universaladapter, thereby converting the PCR product into a product comprisingasymmetric adapters at each terminus.

In some embodiments, the PCR product is a whole genome amplification(WGA) product.

In any of the methods disclosed herein, it is contemplated that thetarget loci chosen for multiplexed amplification correspond to any of avariety of applications, including but not limited to oncology specifictargets, drug resistance specific targets, targets for inheriteddisease, targets from infectious pathogens, targets for pathogen hosts,species-specific targets, and any clinically actionable targets.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is exemplary of alternative existing NGS adapter designsincluding: a fill-in adapter (blunt-ended or with T-overhang) with 3′and 5′ hydroxyls; a Y-adapter (with T-overhang) with 3′ hydroxyl and 5′phosphate; and a stem-loop adapter (blunt-ended or with T-overhang) with3′ hydroxyl and 5′hydroxyl or phosphate.

FIG. 2 is exemplary of alternative existing adapter ligationchemistries. Panel A depicts an adaptor ligation chemistry for use withfill-in adaptors while panel B depicts adaptor ligation chemistry for aY-adaptor.

FIG. 3 depicts the structural features of the 3′ and 5′ adapters of thepresent application.

FIG. 4 depicts one embodiment of the present methods for ligation of the3′ adapter and the 5′ adaptor by nick-translation. The steps include:1—substrate molecule dephosphorylation; 2—substrate moleculepolishing/blunt end generation; 3—3′ adapter ligation; 4—partialdegradation of the 3′ adapter and annealing of the 5′ adapter;5—polymerase extension of the 5′ adapter by nick-translation; and6—ligation of the extended 5′ adapter to the exposed 5′ phosphate of theDNA substrate.

FIG. 5 depicts another embodiment of the present methods for ligation ofthe 3′ adaptor and the 5′ adapter by displacement-cleavage. The stepsinclude: 1—substrate molecule dephosphorylation; 2—substrate moleculeend polishing/blunt end generation; 3—3′ adapter ligation; 4—partialdegradation of the 3′ adapter and annealing of the 5′ adapter;5—displacement of the 5′ base(s) of the DNA fragment and annealing ofthe 3′ base(s) of the 5′ adapter; 6—cleavage of the displaced 5′ base(s)of DNA by a 5′-flap endonuclease; and 7—ligation of the 3′ end of the 5′adapter to the exposed 5′ phosphate of the substrate DNA.

FIG. 6A depicts 5′-adapter attachment by coupledannealing-nick-translation-ligation. This is achieved in two incubationswhere the first incubation is a 3′-adapter attachment, and the secondincubation combines three reactions that occur sequentially: annealingof the 5′-adapter; 5′-adapter extension by DNA polymerase withnick-translation activity (excision of damaged 5′ terminus of substrateDNA); and ligation of the 5′-adapter to the exposed 5′-phosphate of thesubstrate DNA.

FIG. 6B depicts 5′-adapter attachment by coupled annealing-baseexcision-ligation. This is achieved in two incubations where the firstincubation is a 3′-adapter attachment, and the second incubationcombines three reactions that occur sequentially: annealing of the5′-adapter with one or several random bases at the 3′-end anddisplacement of one or several terminal 5′-bases of substrate DNA;cleavage of displaced 5-bases by 5′-flap endonuclease (excision ofdamaged 5′ terminus of substrate DNA); and ligation of the 5′-adapter tothe exposed 5′-phosphate of the substrate DNA.

FIG. 7A provides for an initial fragmentation step resulting ingeneration of a single-stranded 3′ overhang.

FIG. 7B provides four alternative approaches for enzymatically adding a3′-adapter overhang sequence: 1—conventional ligation using T4 DNAligase; 2—single-strand DNA (RNA) ligase; 3—conventional homopolymertailing with terminal transferase; and 4—controlled tailing andsimultaneous adapter ligation using terminal transferase, DNA ligase andattenuator-adapter molecule. See International patent application numberPCT/US13/31104, filed Mar. 13, 2013, incorporated by reference in itsentirety. Alternatively, DNA fragmentation or other processing canresult in pre-existing DNA ends with 3′-overhangs sufficient for 5′adapter annealing.

FIG. 8A depicts steps (I)-(III) for annealing a 5′ adaptor to a nucleicacid substrate. The steps include: I—binding after degradation of thesecond oligonucleotide that was previously annealed to the 3′-adapter;II—competitive displacement of the second oligonucleotide that waspreviously annealed to the 3′-adapter; and III—binding to the upstreamregion of the 3′-adapter (followed by limited nick-translation anddegradation of the second oligonucleotide that was previously annealedto the 3′-adapter).

FIG. 8B depicts additional steps (IV)-(V) of the annealing method shownin FIG. 8A. The steps include: IV—having the 5′-adapter pre-annealed tothe upstream region of the 3′-adapter (followed by limitednick-translation and degradation of the second oligonucleotide that waspreviously annealed to the 3′-adapter); and V—having 3′ blocked5′-adapter instead of the 2^(nd) oligonucleotide that is activated bycleavage.

FIGS. 9A, 9B, 9C, and 9D depict various embodiments for ligation of the5′ adapter using single base extension. In FIG. 9A, during Step 1, 3′adapter attachment occurs leaving a nick at the 3′ terminus ofoligonucleotide 2 (due to 5′ OH on substrate); during Step 2, the 3′terminus of oligonucleotide 2 is extended with a ddNTP; and during Step3, partial degradation of the 3′ adapter oligonucleotide 2 by UDG isfollowed by 5′ adapter annealing and attachment by a DNA ligase. In FIG.9B, during Step 1, 3′ adapter attachment occurs leaving a nick at the 3′terminus of oligonucleotide 2 (due to dideoxy 3′ terminus); during Step2, partial degradation of the 3′ adapter oligonucleotide 2 by UDG,annealing of the 5′ primer and its extension with ddNTP mix isperformed; and during Step 3, degradation of the 5′ primer by RNase H,5′ adapter annealing and attachment by a DNA ligase is performed. InFIG. 9C, during Step 1, 3′ adapter attachment occurs leaving a nick atthe 3′ terminus of oligonucleotide 2 (due to 5′ OH on substrate); duringStep 2, 3′ adapter oligonucleotide 2 is extended with a ddNTP mix; andin Step 3, partial degradation of the 3′ adapter oligonucleotide 2 byUDG, 5′ adapter annealing, single base extension with T7 or T4 DNAPolymerase and dNTP mix and attachment by a ligase is performed. In FIG.9D, during Step 1, 3′ adapter attachment is performed, leaving a nick atthe 3′ terminus of oligonucleotide 2 due to the dideoxy terminus; duringStep 2, partial degradation of the 3′ adapter oligonucleotide 2 by UDG,annealing of the 5′ primer and its extension with a ddNTP mix isperformed; and during Step 3, degradation of the 5′ primer by RNase H,5′ adapter annealing, single base extension with T7 or T4 DNA polymeraseand dNTP mix and attachment by a DNA ligase is performed.

FIG. 10A depicts a method of synthesizing an Illumina NGS library Iusing either a nick translation ligation approach (3-5 on left) or aflap endonuclease approach (3-6 on right). The steps for either approachinclude: 1—substrate molecule dephosphorylation and polishing;2—ligation of the 3′ adapter with Illumina sequence P7; and 3—partialdegradation of the 3′ adapter and annealing of the complementary 5′adapter with Illumina sequence P5. For the nick translation approach,the steps further include: 4—polymerase extension of the 5′ adapter bynick-translation; and 5—ligation of the 3′ end of the 5′ adapter to theexposed 5′ phosphate of the DNA substrate. For the flap endonucleaseapproach, the steps further include: 4—displacement of the 5′ base(s) ofthe DNA substrate and annealing of the 3′ base(s) of the 5′ adapter;5—cleavage of the displaced 5′ base(s) of the DNA substrate by a 5′-flapendonuclease; and 6—ligation of the 3′ end of the 5′ adapter to theexposed 5′ phosphate of the DNA substrate. The library is then amplifiedby PCR using primers P5 and P7′.

FIG. 10B depicts an alternative method of synthesizing an Illumina NGSlibrary I using either a nick translation ligation approach (3-5 onleft) or a flap endonuclease approach (3-6 on right). The steps foreither approach include: 1—substrate molecule dephosphorylation andpolishing; 2—ligation of the 3′ adapter with Illumina sequence P5′; and3—partial degradation of the 3′ adapter and annealing of thecomplementary 5′ adapter with Illumina sequence P7′. For the nicktranslation approach, the steps further include: 4—polymerase extensionof the 5′ adapter by nick-translation; and 5—ligation of the 3′ end ofthe 5′ adapter to the exposed 5′ phosphate of the DNA substrate. For theflap endonuclease approach, the steps further include: 4—displacement ofthe 5′ base(s) of the DNA substrate and annealing of the 3′ base(s) ofthe 5′ adapter; 5—cleavage of the displaced 5′ base(s) of the DNAsubstrate by a 5′-flap endonuclease; and 6—ligation of the 3′ end of the5′ adapter to the exposed 5′ phosphate of the DNA substrate. The libraryis then amplified by PCR using primers P5 and P7′.

FIG. 11 depicts synthesis of an Illumina NGS library II using thefollowing steps: synthesis of NGS library with truncated adapter P7 byone of two methods described in FIGS. 6A and 6B; library amplificationwith truncated or full length degradable primer P7*; degradation of theincorporated P7* primer followed by annealing and ligation of the 5′adapter P5; and if a truncated degradable primer P7* was used, abridge-ligation of the P7*″ adapter to the truncated adapter P7*′ isperformed to complete full-length adapter P7.

FIG. 12A depicts a synthesis method for an Ion Torrent library performedby the following steps: DNA substrate dephosphorylation and polishing;ligation of the 3′ adapter with sequence A1′-P1′; nick-translationligation or base cleavage ligation of the 3′ end of the 5′ adapter withsequence A to the 5′ end of trimmed DNA; and library amplification byPCR using primers A and P1.

FIG. 12B depicts an alternative synthesis method for an Ion Torrentlibrary performed by the following steps: DNA substratedephosphorylation and polishing; ligation of the 3′ adapter withsequence A′; nick-translation ligation or base cleavage ligation of the3′ end of the 5′ adapter with sequence P1-A1 to the 5′ end of trimmedDNA; and library amplification by PCR using primers A and P1.

FIG. 13A depicts another synthesis method for an Ion Torrent librarywith 96 combinatorial barcode sequences using only 20 adapter sequences.The steps include: DNA end dephosphorylation and polishing (not shown);ligation of the (blunt) 3′-adapter P1_(n) with sequence T′_(n)-L′-P1′and 5′ phosphate group and 3′-blocked complementary oligonucleotide withsequence P1_(tr)-L-T_(n); degradation of the 3′-blocked complementaryoligonucleotide P1_(tr)-L-T_(n); annealing of the 5′-adapter A_(m) withsequence A-t_(m)-L to the linker region L′; extension of the 5′-adapterA_(m) by nick-translation polymerization and ligation of the 3′ end ofthe extended 5′-adapter A_(m) to the 5′ end of DNA; and libraryamplification by PCR using primers A and P1.

FIG. 13B depicts various adapters with combinatorial barcodes includeeight adapters P1_(n) containing barcode sequences T₁, T₂, . . . , T₈and twelve adapters A_(m) containing barcode sequences t₁, t₂, . . . ,t₁₂ providing a library having a combinatorial barcode sequencet_(m)-L-T_(n) with up to 96 barcode combinations.

FIG. 14 depicts a method for enrichment of selected restrictionfragments by 5′ adapter ligation. A restriction DNA fragment is selectedby 5′-adapter ligation followed by PCR amplification. Selection occursby two 5′-adapter-selectors A and B containing sequences a and b thatare identical to the 5′ terminal sequences of the restriction fragment.The method of enrichment involves: DNA digestion with restrictionendonuclease; ligation of the 3′-adapter; partial degradation of the3′-adapter and annealing of the 5′-adapter-selectors; invasion of the5′-adapter-selectors into terminal sequences a and b of the restrictionfragment; cleavage of the displaced terminal sequences a and b by a5′-flap endonuclease; ligation of the 5′-adapter-selectors to the endsof the restriction fragment; and amplification of the selectedrestriction fragment by PCR. The initial digestion and ligation of the3′ adaptor and annealing of the 5′-adaptor selectors can be combinedinto a single incubation reaction with the subsequent steps in singleincubation reaction as well.

FIG. 15A depicts a method for target enrichment by primer extension.Enrichment is performed by 5′ adapter attachment where the 3′ overhangis created by extension of a primer complementary to a target DNA regionon a library with adapters A and B and partial digestion of the 5′domain of adapter A. Biotinylated 5′-adapter is annealed to the3′-overhang of adapter A and then ligated to the 5′ end of adapter Aafter trimming by limited nick-translation. Library fragments containingtarget DNA region are then isolated by affinity capture usingstreptavidin magnetic beads, amplified by PCR and analyzed bysequencing.

FIG. 15B depicts a method for target enrichment by primer extension.Enrichment is performed by 5′ adapter attachment where the 3′ overhangis created by extension of a primer complementary to a target DNA regionon a library with adapters A and B and partial digestion of the 5′domain of adapter A. Biotinylated 5′-adapter is annealed to the3′-overhang of adapter A and then ligated to the 5′ end of adapter Aeither after trimming by invasion-cleavage reaction. Library fragmentscontaining target DNA region are then isolated by affinity capture usingstreptavidin magnetic beads, amplified by PCR and analyzed bysequencing.

FIG. 16 depicts an alternative method of synthesizing a NGS libraryusing either a nick translation ligation approach (4-6 on left) or aflap endonuclease approach (4-7 right). The steps for either approachinclude: 1—substrate molecule dephosphorylation; 2—substrate moleculeend polishing/blunt end generation; 3—ligation of the 3′ adapter withsequence A′; and 4—partial degradation of the 3′ adapter and annealingof the complementary 5′ adapter with sequence A. The steps for the nicktranslation approach further include: 5—polymerase extension of the 5′adapter by nick-translation; and 6—ligation of the 3′ end of the 5′adapter to the exposed 5′ phosphate of the DNA substrate. The steps forthe flap endonuclease approach further include: 5—displacement of the 5′base(s) of DNA and annealing of the 3′ base(s) of the 5′ adapter;6—cleavage of the displaced 5′ base(s) of DNA by a 5′-flap endonuclease;and 7—ligation of the 3′ end of the 5′ adapter to the exposed 5′phosphate of the DNA substrate. The library can be amplified by PCRusing single primer A.

FIG. 17 depicts an alternative method of synthesizing a NGS library.Adapter attachment can create a library of double-stranded DNA fragmentswith covalently linked 3′ and 5′ DNA ends. Library construction isperformed by the following method following the initial steps ofsubstrate molecule dephosphorylation (not shown) and substrate moleculeend polishing/blunt end generation (not shown): Step 1—ligation of thehairpin blunt adapter with phosphorylated 5′ end and blocked(optionally) 3′ end; Step 2—partial degradation of the hairpin adapterto create an extendable 3′ end; and Step 3—nick-translation of the 3′end of the hairpin adapter and its ligation to the exposed 5′ phosphateof the DNA substrate.

FIG. 18 depicts a method of synthesizing a circularized NGS library. Themethod includes the initial steps of substrate moleculedephosphorylation (not shown) and substrate molecule end polishing/bluntend generation (not shown) followed by the following steps: Step1—ligation of adapters with a phosphorylated 5′ end and blocked(optionally) 3′ end and mutually complementary sequences X and X′; Step2—degradation of the non-ligated adapter strands to createsingle-stranded 3′ overhangs; Step 3—non-covalent circularization of DNAby annealing of terminal sequences X and X′ (performed at low DNAconcentration); and Step 4—covalent circularization of DNA bynick-translation ligation reaction.

FIG. 19 provides a comparison of conventional adapter ligation to 3′adapter ligation using FAM-labeled oligonucleotide substrates bypolyacrylamide gel electrophoresis as described in Example 1. Lanes 1-2demonstrate ligation products of a fill-in adapter to a FAM-labeledsubstrate, Lanes 4-11 demonstrate ligation products of 3′ adapters witha FAM-labeled substrate; Lanes 3 and 12 are no ligase controls.

FIG. 20 provides a comparison of conventional adapter ligation to 3′adapter ligation using sheared, ˜150 base pair size-selected genomic DNAsubstrate by polyacrylamide gel electrophoresis, as described in Example2. Each lane tests the effect of different polishing enzymes on theefficiency of conventional (fill-in adapter) ligation or 3′ adapterligation.

FIG. 21A and FIG. 21B depicts optimization for 5′ adapter ligation usinga FAM-labeled oligonucleotide substrate, as described in Example 3. InFIG. 21A, the gel electrophoresis depicts Taq polymerase mediated nicktranslation products when varying the dNTP composition or reactiontemperature from 30-50° C. In FIG. 21B, the gel electrophoresis depictsTaq polymerase mediated nick translation when varying the temperaturefrom 50-60° C.

FIG. 22 provides analysis of dNTP composition and ligase effects on 5′adapter nick translation and ligation, as described in Example 4. Thepolyacrylamide gel electrophoresis depicts nick translation and ligationproducts produced from varying the dNTP composition from combinations of3 to 4 nucleotides.

FIG. 23A provides data related to a coupled nick translation-ligationreaction with thermo stable enzymes, as described in Example 5. Thepolyacrylamide gel electrophoresis depicts nick translation ligationproducts produced from varying the dNTP composition from combinations of1-3 nucleotides and varying the reaction temperature when usingthermostable polymerase and ligase.

FIG. 23B provides data related to a coupled nick translation-ligationreaction with thermo stable enzymes, as described in Example 5. Thepolyacrylamide gel electrophoresis depicts nick translation ligationproducts produced from varying the number of units of thermostableligase in the reaction.

FIG. 24 provides data related to a coupleddisplacement-cleavage-ligation reaction, as described in Example 6. Thepolyacrylamide gel electrophoresis depictsdisplacement-cleavage-ligation products when using either E. coli DNAligase (upper panel) or Taq DNA ligase (bottom panel).

FIG. 25 provides data related to a coupleddisplacement-cleavage-ligation reaction with either “N”universal/degenerate or “T” substrate-specific 5′ adapter 3′ overhang,as described in Example 7. The polyacrylamide gel electrophoresisdepicts the effect on 5′ flap endonuclease cleavage mediated ligationwhen the 5′ adapter 3′ terminal overhang is a sequence specific match“T” or if it is composed of a degenerate non-sequence-specific “N”,under varying temperature conditions (panel A), or when theconcentration of 5′ adapter “N” is varied (panel B).

FIG. 26 provides data related to a coupled nick-translation-ligationreaction using DNA polymerase I, as described in Example 8. Thepolyacrylamide gel electrophoresis depicts nick translation mediatedligation products when using either Taq DNA polymerase at 40° C. or DNAPolymerase I at 14-18° C. with 1-5 enzyme units.

FIG. 27 demonstrates polishing is required for blunt ligation ofphysically sheared DNA and dephosphorylation prevents the formation ofchimeric ligation products, as described in Example 9. Thepolyacrylamide gel electrophoresis depicts the ligation productsfollowing treatment with reactions A-D on size-selected sheared DNA,wherein reactions A and D contain polishing enzymes, and reactions B andC contain a phosphatase.

FIG. 28A demonstrates increased NGS Library yield when using 5′ basetrimming coupled to adapter ligation, as described in Example 10. Thequantified library yields obtained from varying library prep conditionsas indicated are depicted in the table.

FIG. 28B relates to the libraries constructed in Example 10 which usedsize-selected sheared DNA so library products could be easily visualizedby polyacrylamide gel electrophoresis, as depicted by the 3′ and 5′adapter ligation products.

FIG. 29A demonstrates the utility of the reactions presented in theirexemplary application to NGS library construction. Libraries wereconstructed from sheared E. coli DNA and sequenced in order todemonstrate the superior evenness of coverage obtained over a wide basecomposition of the genome. The summary of metrics is listed for theFastQC report of sequence metrics from the Illumina MiSeq run.

FIG. 29B depicts FastQC basic statistics from the report

FIG. 29C depicts per base sequence quality of the FastQC report

FIG. 29D depicts per base sequence content from the FastQC report

FIG. 29E depicts per base GC content from the FastQC report

FIG. 29F depicts per sequence GC content from the FastQC report

FIG. 29G depicts sequence duplication levels from the FastQC report

FIG. 29H depicts a Picard CollectGcBiasMetrics plot where the librarycoverage is plotted relative to the base composition of the E. coligenome

FIG. 30 depicts the sequence and structure of exemplary oligonucleotideadapters described in Example 1. The 12-900/13-426 oligonucleotideduplex is the fill-in adapter; the 13-340/13-559 oligonucleotide 1 and 2duplex is the 3′ adapter option 1 with a blocking 3′ deoxythymidine baseat the 3′ terminus of 13-559; and the 13-340/13-558 oligonucleotide 1and 2 duplex is the 3′ adapter option 2 where there is a phosphate groupat the 3′ terminus of 13-558.

FIG. 31 depicts FAM substrate molecules used in Example 1. The13-562/13-563 duplex is a substrate where the FAM group labels ligationto the 5′ phosphate of the substrate; the 13-561/13-564 duplex is asubstrate where the FAM group labels ligation to the 3′ OH of thesubstrate and where the corresponding 5′ terminus of the substrate has aphosphate; the 13-560/13-564 duplex is a substrate where the FAM grouplabels ligation to the 3′ OH of the substrate and where thecorresponding 5′ terminus of the substrate lacks a phosphate.

FIG. 32 depicts the structure of adapters as described in Example 2. The13-489/13-426 oligonucleotide duplex is a fill-in adapter; the13-340/13-559 oligonucleotide 1 and 2 duplex is a 3′ adapter option 1containing a blocking 3′ deoxythymidine base at the 3′ terminus of13-559.

FIG. 33 depicts the oligonucleotide construct system as described inExamples 3, 4, 5, and 8. The 5′ adapter oligonucleotide for nicktranslation is 13-144 (34 bases) in bold type; the FAM oligonucleotidesubstrate is 13-581 (35 bases) in italics type; the oligonucleotidetemplate is 13-582 (47 bases) in standard type.

FIG. 34 depicts the oligonucleotide construct system as described inExample 6, where the construct can exist as shown above (5′ adapter with3′ flap) or the construct can exist as shown below (substrate with 5′flap). The 5′ adapter oligonucleotide for displacement cleavage is13-156 (35 bases) in bold type; the FAM oligonucleotide substrate is13-581 (35 bases) in italics type; and the oligonucleotide template is13-582 (47 bases) in standard type.

FIG. 35 depicts the oligonucleotide construct system as described inExample 7, where the construct can exist as shown above (5′ adapter with3′ flap) or the construct can exist as shown below (substrate with 5′flap). The 5′ adapter oligonucleotide for displacement cleavage “T” is13-607 in bold type; the FAM oligonucleotide substrate is 13-581 (35bases) in italics type; the oligonucleotide template is 13-582 (47bases) in standard type.

FIG. 36 depicts the oligonucleotide construct system as described inExample 7, where the construct can exist as shown above (5′ adapter with3′ flap) or the construct can exist as shown below (substrate with 5′flap). The 5′ adapter oligonucleotide for displacement cleavage is13-596 (65 bases) in bold type; the FAM oligonucleotide substrate is13-581 (35 bases) in italics type; and the oligonucleotide template is13-582 (47 bases) in standard type.

FIG. 37 depicts the structure of P7 and P5 adapters as described inExample 10, where the P7 adapter is depicted above comprising a 3′adapter where the 1^(st) oligonucleotide is 13-501 annealed to the2^(nd) oligonucleotide 13-712; and where two P5 adapters are depictedbelow, the first comprising an oligonucleotide for nick translation(13-489) and the second comprising an oligonucleotide for displacementcleavage with a 3′ terminal N base (13-595).

FIG. 38 depicts the structure of P7 and P5 adapters as described inExample 11, where the P7 adapter is depicted above comprising a 3′adapter where the 1^(st) oligonucleotide is 13-501 annealed to the2^(nd) oligonucleotide 13-712; and where a P5 adapter is depicted below,an oligonucleotide for nick translation (13-489).

FIG. 39 depicts two workflows for the amplicon NGS library constructionmethod, where the diagram on the left depicts a 3 step amplicon librarysynthesis using a two-step PCR followed by a 1 step attachment of NGSadapters; and where the diagram on the right depicts a 2 step ampliconlibrary synthesis using a single step PCR followed by a 1 stepattachment of NGS adapters.

FIG. 40 depicts the first amplicon library workflow where themultiplexed PCR is divided by a purification step. Step 1a is a firstPCR cycle: annealing and extension of the reverse primer comprisingtarget-specific sequence T1 at the 3′ end, universal sequence A1 at the5′ end, and degenerate sequence N between them (UI). Step 1b is a secondPCR cycle: annealing and extension of the forward primer comprisingtarget-specific sequence T1 at the 3′ end, universal sequence A1 at the5′ end and degenerate sequence N (UI) between them and creation of anamplicon with universal sequences A1 and A1′ at the 5′ and 3′ end,respectively. Step 1c is optional, where more PCR cycles with reverseand forward primers to increase the number of produced amplicons isperformed; Optionally, a purification (SPRI beads or a spin column) orExonuclease I treatment is performed; During Step 2, multiple PCR cyclesare performed with a universal primer comprising universal sequence A1,with cleaveable bases such as deoxyuridine, deoxyinosine or RNA, then apurification step of SPRI beads or a spin column is performed.

FIG. 41 depicts the second amplicon workflow where the multiplexed PCRis performed as a single step. Step 1a is a first PCR cycle: annealingand extension of the reverse primer comprising target-specific sequenceT1 at the 3′ end, universal sequence A1 at the 5′ end, and degeneratesequence N between them (UI). Step 1b is a second PCR cycle: annealingand extension of the forward primer comprising target-specific sequenceT1 at the 3′ end, universal sequence A1 at the 5′ end and degeneratesequence N (UI) between them and creation of an amplicon with universalsequences A1 and A1′ at the 5′ and 3′ end, respectively; during Step 1c,multiple PCR cycles are performed with a universal primer comprisinguniversal sequence A1, with cleaveable bases such as deoxyuridine,deoxyinosine or RNA, then a purification step of SPRI beads or a spincolumn is performed.

FIG. 42 depicts the final step to amplicon library synthesis: 1-step NGSadapter attachment of adapter A and B to each amplicon, where Adapter Bis a 5′ adapter and Adapter A is a truncated 3′ adapter. In Step 1a,degradation of the universal 5′ sequence A1 occurs by UDG, endonucleaseV, or RNase H if the cleavable bases within sequence A1 arecorrespondingly deoxyuridines, deoxyinosines or RNA bases, or by 5′exonuclease if sequence A1 has nuclease-resistant phosphorothioatelinkages at the 3′ end; during Step 1b, annealing of adapters A and Boccur; and during Step 1c, ligation of adapters A and B after limitednick-translation reaction or displacement cleavage reaction andsimultaneous linker mediated ligation occur, followed by a purificationstep by SPRI beads or a spin column.

FIG. 43 compares single tube versus two-tube workflow.

FIG. 44A depict amplicon products generated from overlapping primer pairtarget regions.

FIG. 44B further depicts amplicon products at earlier cycles on the leftpanel, where both a maxi-amplicon and a mini-amplicon can form from twooverlapping amplicons 1 and 2; where at the end of the amplification onthe right panel, where current methods would lead to preferentialamplification of the mini-amplicon (top right), the instant inventionprevents amplification of the mini-amplicon (bottom right), insteadfavoring amplification of Amplicon 1, 2 and the maxi-amplicon.

FIG. 45 provides a plot of amplicon coverage over the TP53 coding exons(Example 12).

FIG. 46 provides identification of a somatic mutation in exon 8 of TP53(Example 12).

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention describes a highly efficient method ofadapter ligation to the ends of fragmented double-stranded DNAmolecules. Such DNA molecules are referred to herein as “substratemolecules.” In one aspect, the method comprises a single incubation thatincludes (1) annealing of a 5′ adapter to a pre-existing 3′ overhang ona substrate molecule, preferably a 3′ adapter, (2) removal of a damagedbase from the 5′-termini of the substrate molecules, which enables (3)efficient ligation of the 5′ adapter to the exposed 5′-phosphate of thesubstrate molecules. In another aspect, the method comprises twoincubations, where in the first incubation a 3′ adapter is ligated tothe substrate molecule, and in the second incubation the 5′ adapter isligated to the substrate molecule, as described above (see FIG. 6). Invarious embodiments, the disclosure further provides methods thatcomprise additional steps that occur prior to the one or two ligationsteps, including: (i) a dephosphorylation reaction, (ii) a polishingreaction to excise damaged 3′ termini and generate a blunt end, and(iii) an adenylation reaction; various combinations of the steps arecontemplated by the disclosure, and are discussed in further detailbelow.

In another aspect, disclosure describes a highly efficient method ofmultiplex amplicon NGS library preparation. In one aspect, the methodallows synthesis and amplification of multiple overlapping amplicons ina single tube. In another aspect, it describes a novel, highly efficientmethod of adapter ligation to the ends of PCR amplicons that is free ofchimeric amplicons and adapter-dimers. In one aspect, it allowsincorporation of unique degenerate sequence tags to identify individualamplicons. In another aspect, the method comprises a single incubationthat includes degradation of the 5′ termini of the amplicons followed bysimultaneous ligation of the second adapter B and linker-mediatedligation of the remainder of the 1st adapter A to the substrateamplicons. In various embodiments, the disclosure further providesmethods that comprise additional steps that occur prior to the ligationstep, including: (i) a multiplexed PCR reaction (ii) a purificationstep, and (iii) a universal single primer amplification step.Alternatively, additional steps that occur prior to the ligation stepinclude: (i) a combined multiplex PCR reaction with universal singleprimer amplification, followed by (ii) a purification step. Variousoptions of the steps are contemplated by the disclosure, and arediscussed in further detail below.

The term “reaction conditions” or “standard reaction conditions” as usedherein means conditions according to manufacturer's instructions. It isunderstood that all enzymes herein disclosed are used under standardreaction conditions, unless indicated otherwise. The term “firstpolynucleotide” as used herein is used interchangeably with “3′adapter,” “first adapter,” or “Adapter A” and the term “secondpolynucleotide” as used herein is used interchangeably with “5′adapter,” “second adapter” or “Adapter B.” In certain instances, whenAdapter A is used in reference to IonTorrent™ technology, e.g., FIGS.12-13, it refers to Adapter A as provided by the manufacturer for theIonTorrent™ method, and not “Adapter A” as defined herein.

A “3′ adapter” as used herein ligates to a 3′ end of a substratemolecule, and a “5′ adapter” ligates to a 5′ end of a substratemolecule.

As used herein, a “damaged” 5′ terminus is one that lacks a 5′phosphate.

As used herein, a “processed” substrate molecule is one to which a 5′adapter has been attached.

As used herein, a “high fidelity polymerase” is one that possesses 3′-5′exonuclease (i.e., proofreading) activity.

The term “tolerant,” as used herein, refers to a property of apolymerase that can extend through a template containing a cleavablebase (e.g., uracil, inosine, and RNA).

As used herein, the term “asymmetric” refers to a double strandedmolecule with both adapters at both termini instead of a single adapterat both termini. Thus, the asymmetry arises from the fact that bothadapters are largely non-complementary to each other and have singlestranded portions.

As used herein, a “universal primer” is an oligonucleotide used in anamplification reaction to incorporate a universal adapter sequence. A“universal adapter” as used herein is a portion of the amplificationproduct that corresponds to the universal primer sequence and itsreverse complement.

It will be understood that a modification that decreases the bindingstability of two nucleic acids includes, but is not limited to anucleotide mismatch, a deoxyinosine, an inosine or a universal base.

It will also be understood that a modification that increases thebinding stability of two nucleic acids includes, but is not limited to alocked nucleic acid (LNA), spermine and spermidine or other polyamines,and cytosine methylation.

As used herein, the term “universal base” is one that can base pair withall four naturally occurring bases without hydrogen bonding and is lessdestabilizing than a mismatch, and includes but is not limited to 5′nitroindole.

A “molecular identification tag” as used herein is anywhere between 4and 16 bases in length where the optimal length is between 8 and 12degenerate N bases.

Substrate Molecule

It is contemplated that a substrate molecule is obtained from anaturally occurring source or it can be synthetic. The naturallyoccurring sources include but are not limited to genomic DNA, cDNA, DNAproduced by whole genome amplification, primer extension productscomprising at least one double-stranded terminus, and a PCR amplicon.The naturally occurring source is, in various embodiments, a prokaryoticsource or a eukaryotic source. For example and without limitation, thesource can be a human, mouse, virus, plant or bacteria or a mixturecomprising a plurality of genomes.

As used herein, an “amplicon” is understood to mean a portion of apolynucleotide that has been synthesized using amplification techniques.

If the source of the substrate molecule is genomic DNA, it iscontemplated that in some embodiments the genomic DNA is fragmented.Fragmenting of genomic DNA is a general procedure known to those ofskill in the art and is performed, for example and without limitation invitro by shearing (nebulizing) the DNA, cleaving the DNA with anendonuclease, sonicating the DNA, by heating the DNA, by irradiation ofDNA using alpha, beta, gamma or other radioactive sources, by light, bychemical cleavage of DNA in the presence of metal ions, by radicalcleavage and combinations thereof. Fragmenting of genomic DNA can alsooccur in vivo, for example and without limitation due to apoptosis,radiation and/or exposure to asbestos. According to the methods providedherein, a population of substrate molecules is not required to be of auniform size. Thus, the methods of the disclosure are effective for usewith a population of differently-sized substrate polynucleotidefragments.

The substrate molecule, as disclosed herein, is at least partiallydouble stranded and comprises a 3′ overhang (see FIG. 7a ), a blunt end,a 3′ recessed end, or a free 3′ hydroxyl group. The length of anoverhang or recessed end of a substrate polynucleotide can be varied. Invarious aspects, the length of an overhang or recessed end of asubstrate molecule is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 or more nucleotides in length. In furtherembodiments, the length of an overhang or recessed end of a substratemolecule is at least 1, at least 2, at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19 or at least 20 nucleotides in length.In still further embodiments, the length of an overhang or recessed endof a substrate molecule is from about 1 to about 5, or from about 1 toabout 10, or from about 1 to about 15, or from about 1 to about 20nucleotides in length. A population of substrate molecules, in variousaspects, includes those wherein more than one of the above-mentionedtypes of substrate molecules are present in a single reaction. Thedisclosure also contemplates that the substrate molecule is at leastpartially single stranded. Aspects of the disclosure in which thesubstrate molecule is single stranded involve the use of a singlestranded ligase enzyme.

Some applications of the current invention involve attachment of adaptersequences not to original or native double stranded DNA substratemolecules but to a double stranded DNA produced by primer extensionsynthesis. One example of such an application is a DNA library producedby (a) attachment of an oligonucleotide comprising a primer-bindingsequence to the 3′ end of single-stranded or double-stranded DNA toenable primer extension, (b) extension of the primer annealed to theoligonucleotide, and (c) attachment of the 3′ and 5′ adapters to thedouble-stranded DNA ends produced by the primer-extension.

The length of either a double-stranded portion or a single-strandedportion of a substrate molecule is contemplated to be between about 3and about 1×10⁶ nucleotides. In some aspects, the length of thesubstrate molecule is between about 10 and about 3000 nucleotides, orbetween about 40 and about 2000 nucleotides, or between about 50 andabout 1000 nucleotides, or between about 100 and about 500 nucleotides,or between about 1000 and about 5000 nucleotides, or between about10,000 and 50,000 nucleotides, or between about 100,000 and 1×106nucleotides. In further aspects, the length of the substrate molecule isat least 3 and up to about 50, 100 or 1000 nucleotides; or at least 10and up to about 50, 100 or 1000 nucleotides; or at least 100 and up toabout 1000, 5000 or 10000 nucleotides; or at least 1000 and up to about10000, 20000 and 50000; or at least 10000 and up to about 20000, 50000and 100,000 nucleotides; or at least 20000 and up to about 100,000,200,000 or 500,000 nucleotides; or at least 200,000 and up to about500,000, 700,000 or 1,000,000 nucleotides. In various aspects, thelength of the substrate molecule is about 6, about 7, about 8, about 9,about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23,about 24, about 25, about 26, about 27, about 28, about 29, about 30,about 31, about 32, about 33, about 34, about 35, about 36, about 37,about 38, about 39, about 40, about 41, about 42, about 43, about 44,about 45, about 46, about 47, about 48, about 49, about 50, about 51,about 52, about 53, about 54, about 55, about 56, about 57, about 58,about 59, about 60, about 61, about 62, about 63, about 64, about 65,about 66, about 67, about 68, about 69, about 70, about 71, about 72,about 73, about 74, about 75, about 76, about 77, about 78, about 79,about 80, about 81, about 82, about 83, about 84, about 85, about 86,about 87, about 88, about 89, about 90, about 91, about 92, about 93,about 94, about 95, about 96, about 97, about 98, about 99, about 100,about 110, about 120, about 130, about 140, about 150, about 160, about170, about 180, about 190, about 200, about 210, about 220, about 230,about 240, about 250, about 260, about 270, about 280, about 290, about300, about 310, about 320, about 330, about 340, about 350, about 360,about 370, about 380, about 390, about 400, about 410, about 420, about430, about 440, about 450, about 460, about 470, about 480, about 490,about 500, about 510, about 520, about 530, about 540, about 550, about560, about 570, about 580, about 590, about 600, about 610, about 620,about 630, about 640, about 650, about 660, about 670, about 680, about690, about 700, about 710, about 720, about 730, about 740, about 750,about 760, about 770, about 780, about 790, about 800, about 810, about820, about 830, about 840, about 850, about 860, about 870, about 880,about 890, about 900, about 910, about 920, about 930, about 940, about950, about 960, about 970, about 980, about 990, about 1000, about 1100,about 1200, about 1300, about 1400, about 1500, about 1600, about 1700,about 1800, about 1900, about 2000, about 2100, about 2200, about 2300,about 2400, about 2500, about 2600, about 2700, about 2800, about 2900,about 3000, about 3100, about 3200, about 3300, about 3400, about 3500,about 3600, about 3700, about 3800, about 3900, about 4000, about 4100,about 4200, about 4300, about 4400, about 4500, about 4600, about 4700,about 4800, about 4900, about 5000, 10,000, 15,000, 20,000, 50,000,100,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000,500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000,900,000, 950,000, 1,000,000 or more nucleotides.

Amplicon Molecules

As used herein, an “amplicon” is understood to mean a portion of apolynucleotide that has been synthesized using amplification techniques.

The length of an amplicon is contemplated to be between about 10 bp to175 bp, where the desired amplicon size is significantly shorter thancirculating cell-free DNA fragments (˜165 bp) and small enough in sizeas to not span formalin-induced cross linked DNA from preserved samples,ideally <150 bp in length. It is contemplated the amplicon can be 15 bp,20 bp, 25 bp, 30 bp, 35 bp, 40 bp, 45 bp, 50 bp, 51 bp, 52 bp, 53 bp, 54bp, 55 bp, 56 bp, 57 bp, 58 bp, 59 bp, 60 bp, 61 bp, 62 bp, 63 bp, 64bp, 65 bp, 66 bp, 67 bp, 68 bp, 69 bp, 70 bp, 71 bp, 72 bp, 73 bp, 74bp, 75 bp, 76 bp, 77 bp, 78 bp, 79 bp, 80 bp, 81 bp, 82 bp, 83 bp, 84bp, 85 bp, 86 bp, 87 bp, 88 bp, 89 bp, 90 bp, 91 bp, 92 bp, 93 bp, 94bp, 95 bp, 96 bp, 97 bp, 98 bp, 99 bp, 100 bp, 101 bp, 102 bp, 103 bp,104 bp, 105 bp, 106 bp, 107 bp, 108 bp, 109 bp, 110 bp, lllbp, 112 bp,113 bp, 114 bp, 115 bp, 116 bp, 117 bp, 118 bp, 119 bp, 120 bp, 121 bp,122 bp, 123 bp, 124 bp, 125 bp, 126 bp, 127 bp, 128 bp, 129 bp, 130 bp,131 bp, 132 bp, 133 bp, 134 bp, 135 bp, 136 bp, 137 bp, 138 bp, 139 bp,140 bp, 141 bp, 142 bp, 143 bp, 144 bp, 145 bp, 146 bp, 147 bp, 148 bp,149 bp, 150 bp, 151 bp, 152 bp, 153 bp, 154 bp, 155 bp, 156 bp, 157 bp,158 bp, 159 bp, 160 bp, 161 bp, 162 bp, 163 bp, 164 bp, 165 bp, 166 bp,167 bp, 168 bp, 169 bp, 170 bp, 171 bp, 172 bp, 173 bp, 174 bp, 175 bpor more in length.

Alternatively, for longer reads, particularly for long read sequencetechnologies capable of providing multi-kilobase reads that providehaplotyping information or span repetitive or other difficult sequences(PacBio), amplicon length is contemplated to be between 150 bp to150,000 bp or more in length, when high molecular weight DNA is utilizedas the input DNA for the amplification reaction. It is contemplated theamplicon can be 150 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp,800 bp, 900 bp, 1,000 bp, 2,000 bp, 3,000 bp, 4,000 bp, 5,000 bp, 6,000bp, 7,000 bp, 8,000 bp, 9,000 bp, 10,000 bp, 11,000 bp, 12,000 bp,13,000 bp, 14,000 bp, 15,000 bp, 16,000 bp, 17,000 bp, 18,000 bp, 19,000bp, 20,000 bp, 30,000 bp, 40,000 bp, 50,000 bp, 100,000 bp, 150,000 bpor more in length.

In any of the methods disclosed herein, it is contemplated that thetarget loci chosen for multiplexed amplification correspond to any of avariety of applications, including but not limited to oncology specifictargets, drug resistance specific targets, drug metabolism andabsorption targets (e.g. CYP2D6), targets for inherited disease (e.g.cystic fibrosis CFTR gene, Lynch syndrome MLH1, MSH2, MSH6, PMS2 andEPCAM genes) targets from infectious pathogens, targets for pathogenhost loci, species-specific targets, and any clinically actionabletargets. In one aspect, the target loci are chosen from a set ofoncology targets including but not limited to BRAF, KRAS, EGFR, KIT,HRAS, NRAS, MET, RET, GNA11, GNAQ, NOTCH1, ALK, PIK3CA, JAK2, AKT1,DNMT3A, IDH2, ERBB2 and TP53. In another aspect, the oncology targetsinclude 400-600 genes, including but not limited to the following subsetof genes: ACURL1, AKT1, APC, APEX1, AR, ATM, ATP11B, BAP1, BCL2L1, BCL9,BIRC2, BIRC3, BRCA1, BRCA2, CCND1, CCNE1, CD274, CD44, CDH1, CDK4, CDK6,CDKN2A, CSNK2A1, DCON1D1, EGFR, ERBB2, FBXW7, FGFR1, FGFR2, FGFR3,FGFR4, FLT3, GAS6, GATA3, IGF1R, IL6, KIT, KRAS, MCL1, MDM2, MET, MSH2,MYC, MYCL, MYCN, MYO18A, NF1, NF2, NKX2-1, NKX2-8, NOTCH1, PDCD1LG2,PDGFRA, PIK3CA, PIK3R1, PNP, PPARG, PTCH1, PTEN, RB1, RPS6KB1, SMAD4,SMARCB1, SOX2, STK11, TERT, TET2, TIAF1, TP53, TSC1, TSC2, VHL, WT1 andZNF217. In further embodiments, the target loci are chosen from a subsetof genes known to have clinical relevance in oncology, including but notlimited to ABI1, ABL1, ABL2, ACSL3, AF15Q14, AF1Q, AF3p21, AF5q31,AKAP9, AKT1, AKT2, ALDH2, ALK, ALO17, AMER1, APC, ARHGEF12, ARHH,ARID1A, ARID2, ARNT, ASPSCR1, ASXL1, ATF1, ATIC, ATM, ATP1A1, ATP2B3,ATRX, AXIN1, BAP1, BCL10, BCL11A, BCL11B, BCL2, BCL3, BCLS, BCL6, BCL7A,BCL9, BCOR, BCR, BHD, BIRC3, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD3,BRD4, BRIP1, BTG1, BUB1B, C12orf9, C15orf21, C15orf55, C16orf75,C2orf44, CACNA1D, CALR, CAMTA1, CANT1, CARD11, CARS, CASP8, CBFA2T1,CBFA2T3, CBFB, CBL, CBLB, CBLC, CCDC6, CCNBHP1, CCND1, CCND2, CCND3,CCNE1, CD273, CD274, CD74, CD79A, CD79B, CDC73, CDH1, CDH11, CDK12,CDK4, CDK6, CDKN2A, CDKN2C, CDKN2a(p14), CDX2, CEBPA, CEP1, CEP89,CHCHD7, CHEK2, CHIC2, CHN1, CIC, CIITA, CLIP1, CLTC, CLTCL1, CMKOR1,CNOT3, COL1A1, COL2A1, COPEB, COX6C, CREB1, CREB3L1, CREB3L2, CREBBP,CRLF2, CRTC3, CSF3R, CTNNB1, CUX1, CYLD, D10S170, DAXX, DCTN1, DDB2,DDIT3, DDX10, DDX5, DDX6, DEK, DICER1, DNM2, DNMT3A, DUX4, EBF1, ECT2L,EGFR, EIF3E, EIF4A2, ELF4, ELK4, ELKS, ELL, ELN, EML4, EP300, EPS15,ERBB2, ERC1, ERCC2, ERCC3, ERCC4, ERCC5, ERG, ETV1, ETV4, ETV5, ETV6,EVI1, EWSR1, EXT1, EXT2, EZH2, EZR, FACL6, FAM46C, FANCA, FANCC, FANCD2,FANCE, FANCF, FANCG, FAS, FBXO11, FBXW7, FCGR2B, FEV, FGFR1, FGFR1OP,FGFR2, FGFR3, FH, FHIT, FIP1L1, FLI1, FLJ27352, FLT3, FNBP1, FOXA1,FOXL2, FOXO1A, FOXO3A, FOXO4, FOXP1, FSTL3, FUBP1, FUS, FVT1, GAS7,GATA1, GATA2, GATA3, GMPS, GNA11, GNAQ, GNAS, GOLGA5, GOPC, GPC3, GPHN,GRAF, H3F3A, H3F3B, HCMOGT-1, HEAB, HERPUD1, HEY1, HIP1, HIST1H3B,HIST1H4I, HLA-A, HLF, HLXB9, HMGA1, HMGA2, HNRNPA2B1, HOOK3, HOXA11,HOXA13, HOXA9, HOXC11, HOXC13, HOXD11, HOXD13, HRAS, HSPCA, HSPCB, IDH1,IDH2, IGH\, IGK, IGL, IKZFL IL2, IL21R, IL6ST, IL7R, IRF4, IRTA1, ITK,JAKL JAK2, JAK3, JAZFL JUN, KCNJ5, KDM5A, KDM5C, KDM6A, KDR, KIAA1549,KIAA1598, KIF5B, KIT, KLF4, KLK2, KMT2D, KRAS, KTN1, LAF4, LASP1, LCK,LCP1, LCX, LHFP, LIFR, LMNA, LMO1, LMO2, LPP, LRIG3, LSM14A, LYL1, MAF,MAFB, MALAT1, MALT1, MAML2, MAP2K1, MAP2K2, MAP2K4, MAX, MDM2, MDM4,MDS1, MDS2, MECT1, MED12, MEN1, MET, MITF, MKL1, MLF1, MLH1, MLL, MLL3,MLLT1, MLLT10, MLLT2, MLLT3, MLLT4, MLLT6, MLLT7, MN1, MPL, MSF, MSH2,MSH6, MSI2, MSN, MTCP1, MUC1, MUTYH, MYB, MYC, MYCL1, MYCN, MYD88,MYH11, MYH9, MY05A, MYST4, NAB2, NACA, NBS1, NCOA1, NCOA2, NCOA4, NDRG1,NF1, NF2, NFATC2, NFE2L2, NFIB, NFKB2, NIN, NKX2-1, NONO, NOTCH1,NOTCH2, NPM1, NR4A3, NRAS, NRG1, NSD1, NT5C2, NTRK1, NTRK3, NUMA1,NUP214, NUP98, NUTM2A, NUTM2B, OLIG2, OMD, P2RY8, PAFAH1B2, PALB2, PAX3,PAX5, PAX7, PAX8, PBRM1, PBX1, PCM1, PCSK7, PDE4DIP, PDGFB, PDGFRA,PDGFRB, PERI, PHF6, PHOX2B, PICALM, PIK3CA, PIK3R1, PIM1, PLAG1, PLCG1,PML, PMS1, PMS2, PMX1, PNUTL1, POT1, POU2AF1, POU5F1, PPARG, PPFIBP1,PPP2R1A, PRCC, PRDM1, PRDM16, PRF1, PRKAR1A, PSIP1, PTCH1, PTEN, PTPN11,PTPRB, PTPRC, PTPRK, PWWP2A, RAB5EP, RAC1, RAD21, RAD51L1, RAF1, RALGDS,RANBP17, RAP1GD51, RARA, RB1, RBM15, RECQL4, REL, RET, RNF43, ROS1,RPL10, RPL22, RPL5, RPN1, RSPO2, RSPO3, RUNDC2A, RUNX1, RUNXBP2, SBDS,SDC4, SDH5, SDHB, SDHC, SDHD, 42253, SET, SETBP1, SETD2, SF3B1, SFPQ,SFRS3, SH2B3, SH3GL1, SIL, SLC34A2, SLC45A3, SMAD4, SMARCA4, SMARCB1,SMARCE1, SMO, SOCS1, SOX2, SRGAP3, SRSF2, SS18, SS18L1, SSX1, SSX2,SSX4, STAG2, STAT3, STATSB, STAT6, STK11, STL, SUFU, SUZ12, SYK, TAF15,TALI, TAL2, TBL1XR1, TCEA1, TCF1, TCF12, TCF3, TCF7L2, TCL1A, TCL6,TERT, TET2, TFE3, TFEB, TFG, TFPT, TFRC, THRAP3, TIF1, TLX1, TLX3,TMPRSS2, TNFAIP3, TNFRSF14, TNFRSF17, TOP1, TP53, TPM3, TPM4, TPR, TRA,TRAF7, TRB, TRD, TRIM27, TRIM33, TRIP11, TRRAP, TSC1, TSC2, TSHR, TTL,U2AF1, UBRS, USP6, VHL, VTI1A, WAS, WHSC1, WHSC1L1, WIF1, WRN, WT1,WWTR1, XPA, XPC, XPO1, YWHAE, ZCCHC8, ZNF145, ZNF198, ZNF278, ZNF331,ZNF384, ZNF521, ZNF9 and ZRSR2.

In another aspect, the targets are specific to drug resistance loci,including loci conferring resistance to tyrosine kinase inhibitors usedas targeted anti-tumor agents, other targeted loci related to targetedanti-tumor agents, antibiotic resistance loci, and anti-viral resistanceloci.

In another aspect, detection of enteric, blood-borne, CNS, respiratory,sexually transmitted, and urinary tract pathogens including bacteria,fungi, yeasts, viruses, or parasites can be performed. Pathogens causinginfections of the ear, dermis, or eyes could also be detected.Differentiation between pathovars of bacteria or viruses could beconducted as well as genes promoting antibiotic resistance or encodingtoxins.

The types of genetic lesions that can be detected from sequence analysisof the resulting amplicons include SNV (single nucleotide variants),point mutations, transitions, transversions, nonsense mutations,missense mutations, single base insertions and deletions, largerinsertions and deletions that map between a primer pair, knownchromosomal rearrangements such as translocations, gene fusions,deletions, insertions where primer pairs are designed to flank thebreakpoint of such known rearrangements; copy number variations thatinclude amplification events, deletions and loss of heterozygosity(LOH), aneuploidy, uniparental disomies, and other inherited or acquiredchromosomal abnormalities. In addition, if bisulfite conversion isperformed prior to multiplexed PCR and primers are designed to bisulfiteconverted DNA and optionally do not overlap with CpG dinucleotides whichcan result in various modified sequence states making primer design moredifficult, methylation changes can also be detected using the disclosedmethod.

For amplification of the target loci, the optimal length of the 3′target-specific portion of the primer is between 15 and 30 bases but notlimited to this range, where the target-specific portion of the primeris 5 to 50 bases or 10 to 40 bases or any length in between. The desiredTm defined at 2.5 mM Mg²⁺, 50 mM NaCl and 0.25 μM of oligonucleotides is63° C., where variation in Tm among multiplexed primers is not more than±2.5° C. to ensure even amplification under fixed reaction conditions.Desired GC content of the target-specific portion of the primers isideally 50% but can vary between 30% and 70%. The target-specificprimers are designed to avoid overlap with repetitive, non-uniquesequences or common SNP polymorphisms or known mutations for thecondition being assayed, in order to ensure specific, unbiasedamplification from DNA samples from diverse genetic backgrounds.Additionally, target-specific targets and complementary primer designsshould not be subject to secondary structure formation which wouldreduce performance.

The universal primer comprises cleavable bases including but not limitedto deoxyuridine, deoxyinosine or RNA, and can contain one, two, three,four, five or more cleavable bases. Additionally, the target-specificprimers and the universal primer comprise 1, 2, 3, 4 or more nucleaseresistant moieties at their 3′ termini.

Adapter Molecule

The disclosure contemplates the use of a 5′ adapter and a 3′ adapter(see FIG. 3). According to the disclosure, a 3′ adapter is optionallydouble stranded, comprising an “oligonucleotide 1” and an“oligonucleotide 2.” For such a double stranded substrate molecule, anylength of oligonucleotide 1 and oligonucleotide 2 is contemplated aslong as the two oligonucleotides are capable of annealing to each otherunder standard reaction conditions. Thus, the complementarity betweenoligonucleotide 1 and oligonucleotide 2 is such that they can anneal toeach other. In various embodiments, the complementarity is from about70%, 75%, 80%, 85%, 90%, 95% to about 100%, or from about 70%, 75%, 80%,85%, 90%, to about 95%, or from about 70%, 75%, 80%, 85% to about 90%.In specific embodiments, the degree of complementarity betweenoligonucleotide 1 and oligonucleotide 2 is 70%, 75%, 80%, 85%, 90%, 95%,99% or 100%. In further embodiments, oligonucleotide 2 comprises anucleotide that is susceptible to degradation/removal such as an abasicnucleotide or a ribonucleotide. In certain embodiments, oligonucleotide1 and oligonucleotide 2 are different lengths and oligonucleotide 1hybridizes anywhere along the length of oligonucleotide 2.

In further embodiments, the 5′ adapter is single stranded. Inembodiments wherein the 5′ adapter hybridizes to oligonucleotide 1 ofthe 3′ adapter, it is contemplated in further embodiments that suchannealing results in either a nick, gap or in an overlapping base orbases between the 5′ adapter and the substrate molecule (see FIG. 8). Invarious embodiments, the gap or the number of overlapping bases is 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95 or 100 bases in length. In another embodiment whereinthe 3′ adapter is double stranded, following annealing of the 5′ adapterto the 3′ adapter, an enzyme is added to catalyze the “chewing forward”of the 5′ adapter via nick translation to remove oligonucleotide 2. Insome embodiments, the 5′ adapter additionally comprises a random single,double or more N bases at its 3′ terminus that are not complementary tooligonucleotide 1 and which can anneal to the first base(s) of thesubstrate molecule if its 5′ bases are displaced. In other embodiments,the 5′ adapter is a modified polynucleotide. Modified oligonucleotidescontemplated for use are disclosed in United States Patent ApplicationPublication Number 2011/0129832, incorporated by reference in itsentirety. In a specific embodiment, the 5′ adapter comprises a basemodification selected from the group consisting of a locked nucleic acid(LNA) and a peptide nucleic acid (PNA). In certain embodiments, the5′-adapter oligonucleotide is pre-annealed to the 3′-adapter (see FIG.8).

The disclosure also contemplates the use of a universal adapterincorporated by PCR, a single stranded 5′ adapter and the remainder of a3′ adapter that is ligated to one strand of the universal adapter onpartially processed amplicon substrates. According to the disclosure,ligation of the remainder of the 3′ adapter is mediated by a linker. Forthe linker molecule, any length complementary to the universal adapterand the remainder of the 3′ adapter is contemplated as long as the threeoligonucleotides are capable of annealing to each other under standardreaction conditions. Thus, the complementarity is such that they cananneal to each other. In various embodiments, the complementarity isfrom about 70%, 75%, 80%, 85%, 90%, 95% to about 100%, or from about70%, 75%, 80%, 85%, 90%, to about 95%, or from about 70%, 75%, 80%, 85%to about 90%.

In further embodiments, the 5′ adapter is single stranded. Inembodiments wherein the 5′ adapter hybridizes to the 3′ overhang of theuniversal adapter on the amplicon termini, it is contemplated in furtherembodiments that such annealing results in either a nick or gap betweenthe 5′ adapter and the amplicon substrate. In various embodiments, thegap is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 bases in length.

The length of either a universal adapter, 5′ adapter B or remainder ofthe 3′ adapter A is contemplated to be between about 5 and about 200nucleotides. In some aspects, the length of the universal adapter, 5′adapter or the 3′ adapter is between about 5 and about 200 nucleotides,or between about 5 and about 150 nucleotides, or between about 5 andabout 100 nucleotides, or between about 5 and about 50 nucleotides, orbetween about 5 and about 25 nucleotides, or between about 10 and 200nucleotides, or between about 10 and 100 nucleotides. In furtheraspects, the length of the 5′ adapter or the 3′ adapter is at least 5and up to about 50, 100 or 200 nucleotides; or at least 10 and up toabout 50, 100 or 200 nucleotides; or at least 15 and up to about 50,100, or 200 nucleotides; or at least 20 and up to about 50, 100 or 200nucleotides; or at least 30 and up to about 50, 100 or 200 nucleotides;or at least 40 and up to about 50, 100 or 200 nucleotides. In variousaspects, the length of the substrate molecule is about 5, about 6, about7, about 8, about 9, about 10, about 11, about 12, about 13, about 14,about 15, about 16, about 17, about 18, about 19, about 20, about 21,about 22, about 23, about 24, about 25, about 26, about 27, about 28,about 29, about 30, about 31, about 32, about 33, about 34, about 35,about 36, about 37, about 38, about 39, about 40, about 41, about 42,about 43, about 44, about 45, about 46, about 47, about 48, about 49,about 50, about 51, about 52, about 53, about 54, about 55, about 56,about 57, about 58, about 59, about 60, about 61, about 62, about 63,about 64, about 65, about 66, about 67, about 68, about 69, about 70,about 71, about 72, about 73, about 74, about 75, about 76, about 77,about 78, about 79, about 80, about 81, about 82, about 83, about 84,about 85, about 86, about 87, about 88, about 89, about 90, about 91,about 92, about 93, about 94, about 95, about 96, about 97, about 98,about 99, about 100, about 110, about 120, about 130, about 140, about150, about 160, about 170, about 180, about 190, about 200, about 300,about 400, about 500, about 600, about 700, about 800, about 900, about1000, about 1100, about 1200, about 1300, about 1400, about 1500, about1600, about 1700, about 1800, about 1900, about 2000, about 2100, about2200, about 2300, about 2400, about 2500, about 2600, about 2700, about2800, about 2900, about 3000, about 3100, about 3200, about 3300, about3400, about 3500, about 3600, about 3700, about 3800, about 3900, about4000, about 4100, about 4200, about 4300, about 4400, about 4500, about4600, about 4700, about 4800, about 4900, about 5000, about 5100, about5200, about 5300, about 5400, about 5500, about 5600, about 5700, about5800, about 5900, about 6000, about 6100, about 6200, about 6300, about6400, about 6500, about 6600, about 6700, about 6800, about 6900, about7000, about 7100, about 7200, about 7300, about 7400, about 7500, about7600, about 7700, about 7800, about 7900, about 8000, about 8100, about8200, about 8300, about 8400, about 8500, about 8600, about 8700, about8800, about 8900, about 9000, about 9100, about 9200, about 9300, about9400, about 9500, about 9600, about 9700, about 9800, about 9900, about10000, about 10500, about 11000, about 11500, about 12000, about 12500,about 13000, about 13500, about 14000, about 14500, about 15000, about15500, about 16000, about 16500, about 17000, about 17500, about 18000,about 18500, about 19000, about 19500, about 20000, about 20500, about21000, about 21500, about 22000, about 22500, about 23000, about 23500,about 24000, about 24500, about 25000, about 25500, about 26000, about26500, about 27000, about 27500, about 28000, about 28500, about 29000,about 29500, about 30000, about 30500, about 31000, about 31500, about32000, about 32500, about 33000, about 33500, about 34000, about 34500,about 35000, about 35500, about 36000, about 36500, about 37000, about37500, about 38000, about 38500, about 39000, about 39500, about 40000,about 40500, about 41000, about 41500, about 42000, about 42500, about43000, about 43500, about 44000, about 44500, about 45000, about 45500,about 46000, about 46500, about 47000, about 47500, about 48000, about48500, about 49000, about 49500, about 50000, about 60000, about 70000,about 80000, about 90000, about 100000 or more nucleotides in length.

To complete NGS adapter ligation, the universal adapter primeradditionally comprises modified bases and/or linkages that can bedestroyed enzymatically, chemically or physically. Modifications includebut are not limited to dU-bases, deoxyinosine and RNA bases. Annealingof the single-stranded 5′ adapter to the 3′ overhang of the ampliconsoccurs as result of degradation of one strand of the universal adapterthat corresponds to the incorporated universal primer with cleavablebases. In some embodiments, degradation is achieved enzymatically, morespecifically, by using uracil-DNA glycosylase (UDG), or a combination ofUDG and apurinic/apyrimidinic endonuclease if the oligonucleotidecontains deoxyuracil bases, or by endonuclease V if the oligonucleotidecontains deoxyinosine bases. Degradation can also be performed byincubation with RNase H1 or RNase H2 if the incorporated primer containsRNA bases. In some applications, degradation of the incorporated primercan be performed chemically or physically, for example, by light.Alternatively, the 3′ overhang of the amplicon can be produced bylimited exonuclease digestion of the 5′ end of the amplicon. Suchlimited digestion can be achieved enzymatically, more specifically, byusing T7 Gene 6 exonuclease or lambda 5′→3′ exouclease if the primeroligonucleotide contains nuclease-resistant base(s) at the 3′ end,specifically, a base(s) with phosphorothioate linkage. In this case, theexonuclease reaction stops at the modified base and produces a 3′overhang.

Method—Steps

The first three incubations of the method are pre-ligation steps, andinclude (i) dephosphorylation, (ii) polishing and (iii) optionaladenylation. The remaining 2 incubations of the method include (1) 3′adapter ligation, and (2) 5′ adapter ligation which comprises (a) 5′adapter annealing (b) removal of the 5′ base from the substrate moleculeand (c) 5′ adapter ligation (see FIGS. 4-6). In this aspect, the methodhas up to 3 pre-ligation steps and 2 ligation steps. In another aspect,the method has a single ligation step of the 5′ adapter if the substratemolecule comprises a pre-existing 3′ overhang, preferably serving as a3′ adapter (see FIG. 7a ).

Within the amplification reaction, the number of multiplexed cycles islimited to a minimum of 2 or can be performed as 3 cycles, 4 cycles, 5cycles or more, up to N cycles prior to switching to the non-multiplexeduniversal adapter single primer amplification. The number of universalcycles can be varied from 1 cycle to 40 or more cycles, depending on theDNA input and desired library yield. Following multiplex PCRamplification, a purification step is performed, then the simultaneousadapter ligation step is performed.

Pre-Ligation Steps: (I) Dephosphorylation

Prior to adapter ligation, the DNA ends are optionally processed toimprove efficiency of the adapter ligation reaction. DNA end processingin existing methods typically uses two enzymatic reactions: (a)incubation with a proofreading DNA polymerase(s) to polish DNA ends byremoving the 3′-overhangs and filling-in the recessed 3′ ends and (b)incubation with a polynucleotide kinase to add a phosphate group to the5′ termini. When processing DNA ends some methods also adenylateblunt-ended DNA at the 3′ termini by incubation of polished DNA with anon-proofreading DNA polymerase. Adenylation helps to prevent DNAself-ligation and formation of chimeric products. It also minimizesformation of adapter-dimers due to the presence of dT at the 3′ end ofcorresponding adapters. The current invention addresses these issues ina completely different way. Rather than adding a phosphate group to the5′ ends of the DNA fragments, the method of the invention implements anoptional complete removal of the phosphate group from the 5′ ends of theDNA fragments. Dephosphorylation of DNA ends is achieved by incubationof DNA fragments with an enzyme capable of removing a phosphate from aDNA terminus. Examples of enzymes useful in the methods of thedisclosure to remove a 5′ or a 3′ phosphate include, but are not limitedto, any phosphatase enzyme, such as calf intestinal alkalinephosphatase, bacterial alkaline phosphatase, shrimp alkalinephosphatase, Antarctic phosphatase, and placental alkaline phosphatase,each used according to standard conditions.

(ii) Polishing

After removal of the alkaline phosphatase or its inactivation by heat,DNA substrate molecules are optionally subjected to incubation with aproofreading DNA polymerase in the presence of dNTPs to create bluntends. The reactions are performed according to standard conditions.Dephosphorylated and polished DNA fragments are good substrates forattachment of the 3′ adapter but they are poor substrates for DNAfragment concatamer ligation and chimera formation. They are also poorsubstrates for ligation of a conventional adapter.

In some applications of the current invention, 5′ end dephosphorylationby a phosphatase enzyme can be omitted but the addition of an enzymesuch as T4 polynucleotide kinase to the DNA polishing mix is preferablein this case to assure removal of the phosphate group from the 3′termini prior to DNA polishing. Alternatively, the first twopre-ligation reactions described above, dephosphorylation and polishing,can be executed in any order and result in blunt-ended, double-strandedDNA lacking 5′ phosphate groups at their termini.

(iii) Adenylation

The current invention also contemplates the use of adenylation of the 3′terminus of the blunt-end DNA fragments using DNA polymerases withnon-template polymerase activity including but not limited to (exo-)Klenow fragment of DNA polymerase I, and Taq DNA polymerase. Bothalkaline phosphatase treatment and adenylation reduce the propensity ofDNA fragment self-ligation and formation of chimeric library molecules.In the case of including an adenylation step, the 3′ adapter used in thesubsequent step would require a single T overhang.

Ligation Steps (1) 3′ Adapter Ligation, or, Generation of aSingle-Stranded 3′ Overhang on DNA Substrates

The options are depicted in FIG. 7

Option 1a: 3′ Blocked Oligonucleotide 2 as Part of a Double Stranded 3′Adapter (FIG. 7 a)

Existing NGS library preparation protocols rely on ligation between the3′OH group of the adapter and the 5′ phosphate group at the termini ofthe DNA fragments. For this reason, adapters used in conventionalmethods typically have one functional double-stranded end with a 3′hydroxyl group and optional 5′ phosphate group (see FIGS. 1 and 2). Incontrast, the current invention uses a ligation reaction between the 5′phosphate group of the 3′ adapter and the 3′OH group of DNA fragmentswhile leaving a nick between the 3′ terminus of the 3′ adapter and the5′ terminus of the DNA fragments (see FIG. 3). The 3′ adapter has afunctional double-stranded end with a 5′-phosphate group and in thisoption, a 3′ nucleotide that is not competent for ligation (for examplecomprised of a sugar modified base analogs such as 2′,3′ dideoxy base ora 3′-deoxy base). The 3′ adapter is formed by annealing twooligonucleotides: oligonucleotide 1 that has a phosphate group at the 5′end and a blocking group (such as a C3 spacer) at the 3′ end, andoligonucleotide 2 that lacks a phosphate group at the 5′ end andcomprises a non-ligatable base at the 3′ end. Oligonucleotide 2additionally comprises modified bases and/or linkages that can bedestroyed enzymatically, chemically or physically. In most applications,the end of the 3′ adapter that is involved in ligation with thesubstrate molecule is a blunt end. In applications that involveadenylation of DNA fragments, the ligatable end of the 3′ adapter has a3′ overhang containing a 2′,3′ dideoxythymidine or 3′-deoxythymidinebase (or other modifications of the thymine base that block its abilityto form a covalent linkage with the adjacent base). In otherapplications, the functional end of the 3′ adapter could have either a3′ or 5′ overhang containing multiple bases. During incubation with aDNA ligase, the 5′ phosphate of the 3′ adapter becomes ligated to the 3′terminus of the DNA substrate molecules while leaving a nick between the3′ terminus of the 3′ adapter and the 5′ terminus of the DNA substratemolecules. After the reaction is completed, ligated DNA is subjected topurification by spin-column or SPRI bead-based purification to removeexcess adapters and other components of the ligation reaction.

Option 1b: 3′ Hydroxyl Oligonucleotide 2 as Part of a Double Stranded 3′Adapter (FIG. 7 a)

In an alternative method, a 3′-adapter that is lacking a blocked,unligatable base at the 3′ terminus of oligonucleotide 2 can be used.Ligation of a non-blocked oligonucleotide 2 to the substrate moleculewill still be prevented by the lack of 5′ phosphate on the substratemolecule as a result of the dephosphorylation reaction. The advantage ofusing a non-blocked oligonucleotide 2 is that the 3′ end ofoligonucleotide 2 can be extended by a single base using a dideoxynucleotide mix and a DNA polymerase capable nick-translation DNAsynthesis. This enables an alternate method to perform 5′ base excisionfrom the substrate molecule, see subsequent steps described below. Thedisadvantage of using a non-blocked 3′-adapter is the creation ofadapter-dimers during the ligation reaction which reduces adapterconcentration and as a result, may decrease adapter ligation efficiency.Also for this option, oligonucleotide 2 additionally comprises modifiedbases and/or linkages that can be destroyed enzymatically, chemically orphysically.

Option 2: Single Stranded 3′ Adapter (FIG. 7 a)

In the presence of a ligase (DNA or RNA) capable of covalently attachinga single stranded adapter to a double stranded (or single stranded)substrate molecule, oligonucleotide 2 can be omitted from the reaction.

Option 3: Homopolymer 3′ Adapter (FIG. 7 a)

In the presence of a template independent polymerase such as terminaldeoxynucleotidyl transferase (TdT), poly(A) polymerase, poly(U)polymerase or DNA polymerases that lack 3′-exonuclease proofreadingactivity and comprising a nucleotide, a homopolymer or other tail can beincorporated on the 3′ termini of the substrate molecules that can serveas a 3′ adapter sequence.

Option 4: Controlled Tailing and Simultaneous 3′ Adapter Ligation (FIG.7 a)

In the presence of a template independent polymerase such as TdT,nucleotides, and additionally comprising a ligase and anattenuator-adapter molecule, a synthetic tail and defined 3′ adaptersequence can be incorporated on the 3′ termini of the substratemolecules. See International patent application number PCT/US13/31104,filed Mar. 13, 2013, incorporated herein by reference in its entirety.

Option 5: Omit 3′ Adapter Ligation Step (FIG. 7 b)

In the case of substrate molecules that comprise a pre-existing 3′overhang that is naturally occurring or resulting from a previousenzymatic or other treatment, either as a defined or random sequence, aseparate 3′ adapter ligation step is not required and can be omitted,wherein the pre-existing 3′ overhang can serve as the 3′ adapter.

In an alternative embodiment, a phosphatase enzyme with Zinc and otherreaction components can be added to the 3′ adapter ligation reaction atits completion. Performing a phosphatase reaction following 3′ adapterligation is a means of rendering any non-ligated 3′ adapter moleculesincapable of subsequent ligation, which prevents adapter dimers fromforming in subsequent steps when the 5′ adapter is present.

(2) 5′ Adapter Ligation, which is Comprised of Three Steps that Occur ina Single Incubation (I) Annealing of the 5′ Adapter

In the case of single stranded 3′ adapter ligation (option 2),homopolymer addition (option 3) or use of pre-existing 3′ overhang as 3′adapter (option 5), annealing of the 5′ adapter can be performeddirectly without other consideration as there is no oligonucleotide 2 todegrade or displace.

When ligation of a double-stranded 3′-adapter is used to create asingle-stranded 3′ overhang at the ends of double-stranded DNA (options1a, 1b and 4 above), the 5′-adapter can be annealed to the 3′-adapterusing any of five different options, each of which is discussed belowand depicted in FIG. 8:

-   -   i) following degradation of oligonucleotide 2 that was annealed        to the 3′ adapter    -   ii) by competitive displacement of oligonucleotide 2 that was        annealed to the 3′-adapter    -   iii) by annealing the 5′ adapter further 3′ on oligonucleotide 1        relative to the annealing site of oligonucleotide 2, followed by        nick-translation and degradation of oligonucleotide 2    -   iv) by having the 5′ adapter pre-annealed to the 3′ region of        oligonucleotide 1 of the 3′ adapter, followed by        nick-translation and degradation of oligonucleotide 2    -   v) by having the 5′ adapter with a 3′ blocking group        pre-annealed to the 5′ region of oligonucleotide 1 of the 3′        adapter (instead of oligonucleotide 2), followed by enzymatic        excision of the 3′ blocking group

Option i

Oligonucleotide 2 of the 3′ adapter additionally comprises modifiedbases and/or linkages that can be destroyed enzymatically, chemically orphysically. Modifications include but are not limited to dU-bases,deoxyinosine and RNA bases. Annealing of the single-stranded 5′ adapterto the 5′ portion of oligonucleotide 1 of the 3′ adapter occurs asresult of partial degradation of the 3′ adapter, specifically, ofoligonucleotide 2. In some embodiments, degradation of oligonucleotide 2is achieved enzymatically, more specifically, by using uracil-DNAglycosylase (UDG), or a combination of UDG and apurinic/apyrimidinicendonuclease if the second oligonucleotide contains deoxyuracil bases,or by endonuclease V if the second oligonucleotide contains deoxyinosinebases. Degradation of oligonucleotide 2 can also be performed byincubation with RNase H1 or RNase H2 if the second oligonucleotidecontains RNA bases. In some applications, degradation of the secondoligonucleotide can be done chemically or physically, for example, bylight.

Option ii

In some applications, annealing of the 5′ adapter to oligonucleotide 1of the 3′adapter occurs without degradation of oligonucleotide 2. Inthis case, replacement of oligonucleotide 2 with the single-stranded 5′adapter can be facilitated by higher affinity of the 5′ adapter overthat of oligonucleotide 2 either due to increased complementaritybetween oligonucleotide 1 and the 5′ adapter sequence or due to basemodifications within the 5′ adapter that increase its meltingtemperature (for example, LNA bases). Depending on the design of the 5′adapter, annealing to oligonucleotide 1 of the 3′ adapter could eitherresult in a nick or gap between the 3′ end of the 5′ adapter and the 5′end of the DNA substrate molecule, or in overlap of the 3′ and 5′ basesof the 5′ adapter and DNA substrate molecule, correspondingly.

Option iii

In this case, neither degradable modifications or competitivedisplacement of oligonucleotide 2 is used. Instead, the 5′ adapterreplaces oligonucleotide 2 by annealing to the 3′ adapter further 3′ onoligonucleotide 1 relative to the annealing site of oligonucleotide 2,followed by limited nick-translation “chewing forward” which results indegradation or partial degradation of oligonucleotide 2.

Options iv and v

In these cases, the 5′ adapter constitutes a part of the 3′ adapter andit is present during ligation of the 3′ adapter to the DNA substrate. Inoption iv, the 5′ adapter is pre-annealed to the 3′ adapter further 3′on oligonucleotide 1 relative to the annealing site of oligonucleotide 2(similar to option iii). In option v, the 5′ adapter has a blockinggroup at the 3′ end and it is pre-annealed the 3′ adapter instead ofoligonucleotide 2. After ligation of the 3′ adapter, the blocking groupat the 3′ end of the 5′ adapter is removed enzymatically to allow itsextension by a DNA polymerase.

(II) 5′-Base Removal from the Substrate Molecule Resulting in Exposureof a 5′ Phosphate

In this step, creation of a ligation-compatible 5′ terminal phosphategroup on the substrate molecule is achieved by removal of the damaged 5′terminal base of the DNA substrate molecules either by nick-translationof the 5′ adapter oligonucleotide using a DNA polymerase and nucleotides(option i), by a displacement-cleavage reaction using the 5′ adapter anda 5′-flap endonuclease in the absence of nucleotides (option ii), or bysingle dideoxy base extension from oligonucleotide 2 followed bydisplacement-cleavage using a 5′-flap endonuclease in the absence ofnucleotides (option iii). For the third option, 5′ base excision of thesubstrate molecule occurs prior to 5′ adapter annealing, because it isalternately performed using the annealed oligonucleotide 2 instead ofthe 5′ adapter, but is included in this section to simplify descriptionof the method (see FIG. 9).

Option i

Nick-translation DNA synthesis is initiated at the nick or gap betweenthe 3′ end of the 5′ adapter oligonucleotide and the 5′ end of the DNAsubstrate molecules and stops when the ligation reaction seals the nick(see FIGS. 4 and 6 a). The nick-translation reaction can be performed bybut is not limited to DNA polymerases such as DNA polymerase I(holoenzyme), Taq DNA polymerase, Tth DNA polymerase, and Bst DNApolymerase (holoenzyme). Additional enzymes contemplated for useinclude, without limitation, DNA polymerases with 5′-3′ exonucleaseactivity, 5′ flap endonuclease, and a combination of a stranddisplacement polymerase and a 5′ flap endonuclease.

The reaction conditions contemplated for this step include those where(i) both a polymerase with endogenous 5′ exonuclease activity and aligase are active; (ii) a strand displacement polymerase and flapendonuclease polymerase and ligase are active; (iii) a flap endonucleaseand a ligase are active, (iv) simultaneous activity of both athermostable enzyme and a thermolabile enzyme occur; or (v) whereactivity of only thermostable or only thermolabile enzymes can occur. Insome embodiments, conditions (i) and (ii) are each performed with dNTPsfor nick translation. In a specific embodiment, Taq polymerase and E.coli ligase are used at a reaction temperature of 40° C. In variousembodiments, however, a range of reaction temperatures from 10° C. to75° C. are contemplated.

The nick-translation reaction results in removal of one, two or morebases from the 5′ end of the DNA substrate molecules prior to theligation reaction which occurs between the 5′ adapter extension productand the DNA substrate molecule. Nick-translation synthesis can occur inthe presence of all four nucleotides dGTP, dCTP, dTTP and dATP or theirrestricted combinations. Restricted combinations include but are notlimited to three-nucleotide combinations such as dGTP, dCTP and dATP, ordGTP, dCTP and dTTP, or dGTP, dATP and dTTP, or dCTP, dATP and dTTP,two-nucleotide combination such as dGTP and dCTP, or dGTP and dATP, ordGTP and dTTP, or dCTP and dATP, or dCTP and dTTP, or dATP and dTTP orjust one nucleotide such as dGTP, or dCTP, or dATP, or dTTP.

Option ii

The displacement-cleavage reaction does not require dNTPs but requiresthat the 5′ adapter sequence comprises one, two or more random bases atthe 3′ terminus to create an overlap with the substrate molecule, andwhich comprises a plurality of 5′ adapters in the reaction (see FIGS. 5and 6 b). The displacement-cleavage reaction is initiated by annealingof the 5′ adapters, displacement of the 5′ DNA bases of the DNAsubstrate molecule that overlap with the 3′ bases of the 5′ adapters,and cleavage of the displaced bases by a 5′-flap endonuclease. In someembodiments, the 5′ adapter has one random base dN at the 3′ end. Inthis case the overlap involves one base and only a single 5′ base wouldbe removed from the 5′ end of DNA substrate molecules and replaced witha similar base from the 5′ adapter sequence. Efficiency of thedisplacement-cleavage reaction is increased by cycling the temperatureof the reaction between 40° C. and 65° C. to allow 5′ adapters todissociate and re-anneal if its terminal 3′ base is mismatched to the 5′base of the DNA substrate molecule.

Option iii

An alternative embodiment to the 5′ adapter participating in the 5′ baseexcision of the substrate molecules is to instead, in a previous step,have oligonucleotide 2 of the 3′ adapter participate in the 5′ baseexcision of the substrate molecules (see FIG. 9).

In one approach (FIGS. 9a and c ), oligonucleotide 2 of the 3′ adaptercomprises an extendable 3′ terminus and in the presence of a dideoxynucleotide mixture and a polymerase under appropriate conditions, asingle dideoxy base addition occurs which leads to a single base overlapwith the 5′ terminus of the substrate molecules, which induces singlebase displacement-cleavage by an appropriate flap endonuclease orpolymerase that possesses 5′ flap endonuclease activity. Subsequently, a5′ adapter with a random dN base at its 3′ terminus is used (FIG. 9a ),where a nick is formed after binding to the 3′-adapter attached to theend of double stranded DNA. The nick can be sealed by a DNA ligaseresulting in covalent attachment of the 5′adapter to the 5′ terminus ofthe DNA substrate molecule.

Alternatively, a 5′adapter oligonucleotide that lacks a random dN baseat its 3′ terminus can be used (FIG. 9c ), which forms a single base gapafter binding to the 3′-adapter attached to the end of double strandedDNA substrate molecule. The gap can be filled in by a DNA polymeraselacking strand-displacement activity (for example T7 or T4 DNApolymerase) to create a nick that can be in turn sealed by a DNA ligaseresulting in covalent attachment of the 5′adapter to the 5′ end of DNAsubstrate molecule.

In another alternative (see FIGS. 9b and d ), oligonucleotide 2 thatcomprises a blocked 3′ terminus is partially degraded or displaced by aprimer oligonucleotide that becomes extended with a single dideoxy-baseby a DNA polymerase with 5′ flap endonuclease activity resulting inexcision of a single base from the 5′ terminus of DNA. The primeroligonucleotide, in turn, becomes degraded or displaced by the 5′adapter with a random dN base at its 3′ terminus to create a nick thatcan be sealed by a DNA ligase.

(III) Ligation of the 5′ Adapter

Covalent attachment of the 5′ adapter to the substrate molecule involvesligation between the 5′ adapter or its extension product and the exposed5′ phosphate of the substrate molecules. When excision of the 5′ base(s)of DNA substrate molecules is achieved by a nick-translation reaction,the ligation reaction seals the nick between the polymerase-extended 5′adapter and the excised 5′ end of the DNA substrate molecule. Whenexcision of the 5′ base of DNA substrate molecules is achieved throughthe displacement-cleavage reaction, the ligation occurs between theoriginal 5′ adapter oligonucleotide and the excised 5′ end of the DNAsubstrate molecule. The standard conditions with respect to the ligationreaction in this step comprise, in various embodiments, use of any DNAligase that is capable of sealing nicks or gaps in DNA. In oneembodiment, the ligase is E. coli DNA ligase and the reaction occurs inthe temperature interval between 10° C. and 50° C. In some embodiments,the ligase is a thermostable DNA ligase such as Taq DNA ligase, orAmplLigase, and the reaction occurs in the temperature interval between30° C. and 75° C.

In various aspects of the current invention, the three steps (I), (II)and (III) of the 5′ adapter ligation step are performed simultaneouslyin a single incubation by mixing and incubating the 3′-adapted substrateDNA with (i) an optional degradation endonuclease (e.g., UDG,endonuclease V, RNase H, or their combination); (ii) a nick-translationDNA polymerase or a 5′-flap endonuclease; and (iii) a DNA ligase (seeFIG. 6). The incubation is carried out at a constant temperature orusing temperature cycling conditions in the interval 10° C.-75° C. Inother applications, 3′ adapter partial degradation is performedseparately from the downstream reactions.

Construction of NGS Libraries

Synthesis of an Illumina NGS library can be performed using thedisclosed methods. As shown in FIG. 10, an Illumina library can beconstructed using either the nick translation ligation method (leftside) or the displacement cleavage ligation method (right side). Theorder of attachment of the two Illumina adapters is flexible, where inFIG. 10a , Illumina adapter P7 is a 3′ adapter and Illumina adapter P5is a 5′ adapter, whereas in FIG. 10b , Illumina adapter P5 is a 3′adapter and Illumina adapter P7 is a 5′ adapter. The libraries depictedin FIG. 10 can be constructed PCR-free or can be PCR amplified,depending on the amount of input substrate DNA. Alternatively, synthesisof Illumina NGS libraries can be performed using the disclosed methodswhere PCR amplification is required, because the method uses truncatedadapter sequences (see FIG. 11). In this case, either P5 or P7 isintroduced as a truncated adapter (only P7 shown), and followingamplification using a PCR primer that introduces the full-length adaptersequence as well as comprises degradable bases at its 5′ terminus,following degradation of the 5′ portions of the resulting amplicons,either P7 or P5 can be introduced by annealing and ligation. Ifalternatively a truncated degradable primer is used for the PCRamplification, a bridge-ligation of the remainder of the adapter can beperformed to complete the full-length sequence.

The disclosed methods can be used to construct NGS libraries for avariety of sequencing platforms, and another example is presented inFIGS. 12 and 13 where Ion Torrent library construction is depicted. Asshown in FIG. 12, by introducing a partial duplication of the A adaptersequence on the P1 adapter at the insert junction site, subsequentannealing of a 5′ adapter after 3′ adapter ligation can occur. The orderof ligation is flexible, where adapter P1 with a partial duplication ofadapter A can be introduced as a 3′ adapter followed by ligation ofadapter A as a 5′ adapter using either nick translation or displacementcleavage (FIG. 12a ). Alternatively, adapter A can be introduced as a 3′adapter and adapter P1 with a partial duplication of adapter A can be a5′ adapter (FIG. 12b ). Since Ion Torrent sequencing is performed as asingle read from the A adapter, due to the length of the partialduplication of adapter A on the P1 adapter, it will not interfere withsequencing primer annealing or other adapter functions.

Alternatively in FIG. 13, combinatorial barcoding can be introduced toIon Torrent libraries using the disclosed method. During the 3′ adapterligation step, the first portion of the dual combinatorial barcode isintroduced, adjacent to a linker region L that is common to all 20barcodes. After degradation of the 3′ blocked strand that does notligate to the DNA substrate, a 5′ adapter anneals to the common linkerregion L which incorporates the second portion of the dual barcode 5′adjacent to the linker region L. Following nick translation ligation,the resulting library can be amplified with standard Ion Torrent PCRprimers, and when library molecules are sequenced from the A adapterside, the sample identification of each Ion sphere will be read at thebeginning of the read, where 96 possible combinations can be achieved.

Applications for Target Selected NGS Libraries

The disclosed methods can be used to construct NGS libraries wherespecific targets can be selected and enriched, as a way to reducecomplexity and sequencing requirements relative to whole genomesequencing. An example of such an application would be attachment of the3′ adapter and 5′ adapter to randomly fragmented, denatured andprimer-extended DNA substrates, where the primer or plurality of primersanneal to known targeted DNA regions. In this case, only the targetedloci would comprise a double stranded terminus, where non-selected lociwould remain single stranded and adapter ligation would not occur ontheir termini.

In other applications, the 5′ adapter of the current invention can beused to select and enrich a small fraction of DNA fragments with knownterminal sequences. Pre-selected DNA sequences could contain one, two,three or more terminal DNA bases. To achieve such selection the 5′adapter sequence should contain selected invasion bases or basecombinations at the 3′ end. As a result, only DNA fragments withselected terminal sequences will be ligated to the 5′ adapter andamplified. As shown in FIG. 14, use of 5′ adapters with 3′ terminicomplementary to the terminal sequences of selected restrictionfragments can be used to select restriction fragment targets from aplurality of restriction fragments. In another embodiment, use of 5′adapters with 3′ termini comprising CpG dinucleotides would enrich forfragments originating from CpG islands.

Alternatively, target selection can be performed following libraryconstruction using the methods disclosed within (see FIG. 15). If such alibrary is constructed where one adapter comprises degradable bases atits 5′ terminus, following target-specific primer extension and partialdigestion of the degradable portion of the adapter, a biotinylated 5′adapter can be annealed to the resulting 3′ overhang and using eithernick translation ligation (FIG. 15a ) or displacement cleavage ligation(FIG. 15b ), the biotinylated 5′ adapter is covalently attached to onlytargeted DNA substrates and can be subsequently captured usingstreptavidin magnetic beads and then PCR amplified to generatesufficient material for sequencing.

Alternative Adapter Designs and Applications

Several alternative adapter designs and ligation methods using thedisclosed methods are also presented. In FIG. 16, a library isconstructed using a single adapter sequence instead of a pair of adaptersequences. In this example, the same steps are used for substrateprocessing prior to ligation and both 3′ adapter ligation and eithernick translation ligation or displacement cleavage ligation of the 5′adapter, and the resulting library can be PCR amplified using a singleprimer.

In FIG. 17, a method for ligation of single oligonucleotide hairpinadapters is presented, wherein the 5′ terminus of the hairpin adapter isused to perform 3′ adapter ligation to the substrate molecule, andfollowing degradation of the blocked 3′ terminus of the hairpin adapter,the truncated 3′ terminus of the hairpin adapter is used for nicktranslation ligation to the exposed 5′ phosphate of the substratemolecule.

Sometimes it is useful to generate circular DNA libraries, such as anintermediate structure for the construction of mate-pair NGS libraries.As shown in FIG. 18, such a library can be constructed using methods ofthe disclosure. In the first step, 3′ adapter ligation is performedusing mutually complementary adapters X and X′. Following degradation ofthe non-ligated strand, non-covalent DNA circularization can occur bymeans of complementarity of the 3′ overhangs X and X′ on each substratemolecule. To favor unimolecular annealing and reduce concatamerformation, this annealing reaction is performed at an appropriately lowDNA concentration. Following 3′ overhang annealing, nick translationligation can be performed.

Enzymes

Ligases that may be used according to standard reaction conditions topractice the methods of the disclosure include but are not limited to T4DNA ligase, T4 RNA ligase, T3 DNA ligase or T7 DNA ligase, Taq DNAligase, Ampligase, E. coli DNA ligase and E. coli RNA ligase. Thedisclosure contemplates, in various embodiments, reaction conditionsappropriate for a blunt end or a cohesive (“sticky”) end ligation. Thecohesive end, in some embodiments, comprises either a 5′ overhang or a3′ overhang.

Examples of enzymes useful in the methods of the disclosure to remove a5′ or a 3′ phosphate include, but are not limited to, any phosphataseenzyme, such as calf intestinal alkaline phosphatase, bacterial alkalinephosphatase, shrimp alkaline phosphatase, Antarctic phosphatase, andplacental alkaline phosphatase, each used according to standardconditions. Additionally, the phosphatase activity of T4 polynucleotidekinase can be used to remove 3′ phosphate groups.

The polymerase enzymes useful in the practice of the invention includebut are not limited to a DNA polymerase (which can include athermostable DNA polymerase, e.g., a Taq DNA polymerase), RNApolymerase, DNA polymerase I and reverse transcriptase. Non-limitingexamples of enzymes that may be used to practice the present inventioninclude but are not limited to KAPA HiFi and KAPA HiFi Uracil+, VeraSeqUltra DNA Polymerase, VeraSeq 2.0 High Fidelity DNA Polymerase, TakaraPrimeSTAR DNA Polymerase, Agilent Pfu Turbo CX Polymerase, Phusion U DNAPolymerase, Deep VentR™ DNA Polymerase, LongAmp™ Taq DNA Polymerase,Phusion™ High-Fidelity DNA Polymerase, Phusion™ Hot Start High-FidelityDNA Polymerase, Kapa High-Fidelity DNA Polymerase, Q5 High-Fidelity DNAPolymerase, Platinum Pfx High-Fidelity Polymerase, Pfu High-Fidelity DNAPolymerase, Pfu Ultra High-Fidelity DNA Polymerase, KOD High-FidelityDNA Polymerase, iProof High-Fidelity Polymerase, High-Fidelity 2 DNAPolymerase, Velocity High-Fidelity DNA Polymerase, ProofStartHigh-Fidelity DNA Polymerase, Tigo High-Fidelity DNA Polymerase,Accuzyme High-Fidelity DNA Polymerase, VentR® DNA Polymerase, DyNAzyme™II Hot Start DNA Polymerase, Phire™ Hot Start DNA Polymerase, Phusion™Hot Start High-Fidelity DNA Polymerase, Crimson LongAmp™ Taq DNAPolymerase, DyNAzyme™ EXT DNA Polymerase, LongAmp™ Taq DNA Polymerase,Phusion™ High-Fidelity DNA Polymerase, Taq DNA Polymerase with StandardTaq (Mg-free) Buffer, Taq DNA Polymerase with Standard Taq Buffer, TaqDNA Polymerase with ThermoPol II (Mg-free) Buffer, Taq DNA Polymerasewith ThermoPol Buffer, Crimsom Taq™ DNA Polymerase, Crimson Taq™ DNAPolymerase with (Mg-free) Buffer, Phire™ Hot Start DNA Polymerase,VentR® (exo-) DNA Polymerase, Hemo KlenTaq™, Deep VentR™ (exo-) DNAPolymerase, Deep VentR™ DNA Polymerase, DyNAzyme™ EXT DNA Polymerase,Hemo KlenTaq™, LongAmp™ Taq DNA Polymerase, ProtoScript® AMV FirstStrand cDNA Synthesis Kit, ProtoScript® M-MuLV First Strand cDNASynthesis Kit, Bst DNA Polymerase, Full Length, Bst DNA Polymerase,Large Fragment, 9° Nm DNA Polymerase, DyNAzyme™ II Hot Start DNAPolymerase, Hemo KlenTaq™, Sulfolobus DNA Polymerase IV, Therminator™ γDNA Polymerase, Therminator™ DNA Polymerase, Therminator™ II DNAPolymerase, Therminator™ III DNA Polymerase, Bsu DNA Polymerase, LargeFragment, DNA Polymerase I (E. coli), DNA Polymerase I, Large (Klenow)Fragment, Klenow Fragment (3′→5′ exo-), phi29 DNA Polymerase, T4 DNAPolymerase, T7 DNA Polymerase (unmodified), Terminal Transferase,Reverse Transcriptases and RNA Polymerases, E. coli Poly(A) Polymerase,AMV Reverse Transcriptase, M-MuLV Reverse Transcriptase, phi6 RNAPolymerase (RdRP), Poly(U) Polymerase, SP6 RNA Polymerase, and T7 RNAPolymerase.

The enzymes possessing flap endonuclease activity that are useful in thedisclosure include but are not limited to flap endonuclease 1 (FEN1), T5exonuclease, Taq DNA polymerase, Bst polymerase, Tth polymerase, DNApolymerase I and their derivatives.

EXAMPLES Example 1 Comparison of Conventional Adapter Ligation to 3′Adapter Ligation with FAM-Labeled Oligonucleotides

Rationale: Using a FAM-labeled oligonucleotide system, blunt ligationusing fill-in adapters (FIG. 2A) or 3′ adapters (FIG. 3) was tested atdifferent molar ratios of substrate to adapter to examine the effect onligation efficiency and chimera formation.

Materials:

-   -   Fill-in adapter contains oligonucleotides 12-900 and 13-426        (Table 1)    -   3′Adapter; 1^(st) oligonucleotide 13-340 (Table 1)    -   3′Adapter; 2^(nd) oligonucleotide option 1 (with a blocking 3′        deoxythymidine base at the 3′ terminus) 13-559 (Table 1)    -   3′Adapter; 2^(nd) oligonucleotide option 2 (a phosphate group at        the 3′ terminus) 13-558 (Table 1)    -   FAM substrate A composed of oligonucleotides 13-562 and 13-563,        where the FAM group labels ligation to the 5′ Phosphate of the        substrate (Table 1)    -   FAM substrate B composed of oligonucleotides 13-561 and 13-564,        where the FAM group labels ligation to the 3′ OH of the        substrate and where the corresponding 5′ terminus of the        substrate has a phosphate (Table 1)    -   FAM substrate C composed of oligonucleotides 13-560 and 13-564,        where the FAM group labels ligation to the 3′ OH of the        substrate and where the corresponding 5′ terminus of the        substrate lacks a phosphate (Table 1)    -   T4 DNA Ligase (Rapid) (Enzymatics, Cat #L6030-HC-L)    -   10× T4 DNA Ligase Buffer (Enzymatics, Cat #B6030)

Method

Conventional adapter ligation reactions were assembled in a total volumeof 10 μl, comprising 1× T4 DNA Ligase Buffer, 10 pmoles of FAM substrateA, 20 or 200 pmoles of Fill-in adapter, 600 units T4 DNA Ligase (Rapid)or no ligase.

3′ adapter ligation reactions were assembled in a total volume of 10 μl,containing 1× T4 DNA Ligase Buffer, 10 pmoles of FAM substrate B or 10pmoles of FAM substrate C, 20 or 200 pmoles of 3′Adapter option 1 or 20or 200 pmoles of 3′Adapter option 2 and 600 units T4 DNA Ligase (Rapid)or no T4 DNA ligase.

All ligation reactions were performed at 25° C. for 30 minutes. Thetotal ligation reaction volume (10 μl) was mixed with 10 μl of 2×formamide loading buffer (97% formamide, 10 mM EDTA, 0.01% bromophenolblue and 0.01% xylene cyanol), heated at 95° C. for 5 minutes andsubsequently run on a pre-cast 15% polyacrylamide gel, TBE-Urea(Invitrogen, Cat #S11494) in an oven at 65° C., visualized on a Darkreader light box (Clare Chemical Research) and photographed using adigital camera. Subsequently the gel was stained SYBR® Gold nucleic acidgel stain (Invitrogen, Cat #S11494) (not shown).

Results

FAM substrate A was converted into ligation product in the presence ofthe fill-in adapter and T4 DNA ligase (FIG. 19, lanes 1-2). Thisconventional adapter ligation showed some FAM substrate A chimeraformation when a ratio of only 2:1 adapter:substate (FIG. 19, lane 1)was used compared to a ratio of 20:1 (lane 2). No ligation product wasobserved in absence of T4 DNA ligase (FIG. 19, lane 3).

Different scenarios of 3′ adapter ligation were tested in lanes 4 to 12(FIG. 19). Lanes 4 and 5 show ligation reactions between FAM substrate Band 3′ Adapter option 1. At 2:1 (lane 4) or 20:1 (lane 5)adapter:substate ratio, chimeric products of higher molecular weightformed which may or not involve the 3′ Adapter. However, the ligationproduct was more abundant and its formation favored at a ratio of 20:1adapter:substate (lane 5). Lanes 6 and 7 show ligation reactions betweenFAM substrate C and 3′ Adapter option 1. The reaction was favored at aratio of 20:1 adapter:substate (lane 7) and no chimeric products wereobserved. Lanes 8 and 9 show ligation reactions between FAM substrate Band 3′ Adapter option 2. No ligation product was observed, howeverchimeric products were detected. Lanes 10 and 11 show ligation reactionsbetween FAM substrate C and 3′ Adapter option 2. No ligation product wasobserved. No ligation product was observed in absence of T4 DNA ligase(lane 12).

Conclusion

Conventional adapter ligation required a 5′-phosphate on the FAMsubstrate which led to the formation of chimeras if the fill-in adapterswere not in excess. Ligation of the 3′ Adapter was more efficient andwith fewer chimeras when the FAM substrate had a 5′hydroxy group and the3′ Adapter had a blocking 3-deoxythymidine base (option 1) whichprevented ligation between adapter molecules and favored the ligationbetween substrate and adapter. In both cases, the ratio of adapter:substate of 20:1 was favored for ligation product formation.

Example 2 Comparison of Conventional Adapter Ligation to 3′ AdapterLigation with Sheared, Size-Selected Genomic DNA

Rationale: This experiment was performed to test the effect of polishingof physically sheared genomic DNA on the efficiency of conventional or3′ adapter ligation

Materials:

-   -   Fill-in adapter contains oligonucleotides 13-489 and 13-426        (Table 1)    -   3′Adapter; 1^(st) oligonucleotide 13-340 (Table 1) and 2nd        oligonucleotide option 1 (containing a blocking3′ deoxythymidine        base at the 3′ terminus) 13-559 (Table 1)    -   NEBuffer 2 (New England Biolabs, cat #B7002S)    -   100 mM 2′-deoxynucleoside 5′-triphosphate (dNTP) Set, PCR Grade        (Invitrogen (Life technologies), cat #10297-018)    -   Adenosine 5′-Triphosphate (ATP) (New England Biolabs, cat        #P0756S)    -   DNA Polymerase I, Large (Klenow) Fragment (New England Biolabs,        cat #M0210S)    -   T4 DNA polymerase (New England Biolabs, cat #M0203S)    -   T4 Polynucleotide Kinase (New England Biolabs, cat #M0201S)    -   Exonuclease III (E. coli) (New England Biolabs, cat #M0293S)    -   Antarctic Phosphatase (New England Biolabs, cat #M0289S)    -   Antarctic Phosphatase reaction buffer (New England Biolabs, cat        #B0289S)    -   T4 DNA Ligase (Rapid) (Enzymatics, cat #L6030-HC-L)    -   10× T4 DNA Ligase Buffer (Enzymatics, cat #B6030)    -   E. coli genomic DNA ATCC 11303 strain (Affymetrix, cat #14380)    -   M220 Focused-ultrasonicator, (Covaris, cat #PN 500295)    -   Pippin Prep (Sage Science)    -   CDF2010 2% agarose, dye free w/ internal standards (Sage        Science)    -   DNA Clean & Concentrator-5 (Zymo research, cat #D4004)    -   25 bp ladder DNA size marker (Invitrogen (Life technologies),        cat #10488-022)

Method

E. coli genomic (gDNA) was resuspended in DNA suspension buffer(Teknova, cat #T0227) at a concentration of 100 ng/ul. The DNA wasfragmented with the M220 Focused-ultrasonicator to 150 base pairsaverage size. A tight size distribution of fragmented DNA fromapproximately 150 bp to approximately 185 bp was subsequently isolatedon a 2% agarose gel using Pippin Prep.

200 ng of the size-selected DNA was subjected to the activity ofdifferent enzymes. The reactions were assembled in a total volume of 30μl, comprising a final concentration of 1× NEBuffer 2, 100 μM of eachdNTP, 3 units T4 DNA polymerase or 5 units DNA Polymerase I, Large(Klenow) Fragment or 3 units T4 DNA polymerase and 5 units DNAPolymerase I, Large (Klenow) Fragment or 3 units T4 DNA polymerase and 5units DNA Polymerase I, Large (Klenow) Fragment and 1 unit ofExonuclease III. Another reaction was assembled in a total volume of 30μl comprising a final concentration 1× NEBuffer 2, 1 mM ATP, 10 units ofT4 Polynucleotide Kinase. Another reaction was assembled in a totalvolume of 30 μl comprising a final concentration 1× AntarcticPhosphatase reaction buffer and 5 units of Antarctic phosphatase. Acontrol reaction was assembled with 200 ng of the size-selected DNA with1× NEBuffer 2. All reactions were incubated at 37° C. for 30 minutes andthe DNA purified using the DNA Clean & Concentrator-5 columns. DNA waseluted in 30 μl of DNA suspension buffer and divided into 2 tubes of 15μl for subsequent conventional adapter ligation or 3′ adapter ligation.The conventional adapter ligations were assembled in a total volume of30 μl comprising 1× T4 DNA Ligase Buffer, Fill-in adapter containingoligonucleotides 13-489 (220 pmoles) and 13-426 (440 pmoles), and 1200units of T4 DNA Ligase (Rapid). The 3′ adapter ligation reactions wereassembled in a total volume of 30 μl, containing 1× T4 DNA LigaseBuffer, 220 pmoles of 3′ Adapter 1^(st) oligonucleotide, 440 pmoles of3′Adapter 2^(nd) oligonucleotide and 1200 units T4 DNA Ligase (Rapid).All reactions were purified using DNA Clean & Concentrator-5-columns.The DNA was resuspended in 10 μl of DNA suspension buffer and was mixedwith 10 μl of 2× formamide loading buffer (97% formamide, 10 mM EDTA,0.01% bromophenol blue and 0.01% xylene cyanol), heated at 95° C. for 5minutes and subsequently run on a pre-cast 6% polyacrylamide gel,TBE-Urea (Invitrogen, Cat #S11494) in an oven at 65° C. The gel wasstained SYBR® Gold nucleic acid gel stain (Invitrogen, Cat #S11494) andvisualized on a Dark reader light box (Clare Chemical Research) andphotographed using a digital camera.

Results

The conventional adapter ligation reactions (FIG. 20, upper panel) whichrequire a 5′ phosphate on the sheared DNA substrate showed a lowerefficiency than the 3′ adapter ligation which does not (FIG. 20, lowerpanel). The ligation reactions were more efficient after treating DNAwith T4 DNA polymerase alone (lane 3) or in combination with Klenow(lane 7) or Klenow plus Exonuclease III (lane 8) for both types ofligations. Treatment with Klenow, T4 Polynucleotide Kinase or Antarcticphosphatase alone (lanes 4, 5 and 6, respectively) only moderatelyenhanced blunt ligation compared to the non-treated DNA (lane 2). Thetight range distribution fragmented DNA was loaded on lane 9.

Conclusion

Ligation of blunt adapters to sheared DNA highly depends on thepolishing of this DNA. DNA polymerases like T4 DNA polymerase whichpresent a strong 5′ to 3′ exonuclease activity and a 5′ to 3′ polymeraseactivity are well suited for this purpose. The conventional adapterligation reaction depends on the presence of an intact 5′ phosphate onthe substrate's blunt end. However, ligation of the 3′ adapter does not,since the ligation occurs at the 3′ hydroxyl terminus of the fragmentedDNA. Since the 5′ termini of sheared DNA are not enzymatic substratesfor T4 DNA polymerase, this explains why the 3′ adapter was moresuccessfully ligated than the fill-in adapter (lane 3). The combinationof T4 DNA Polymerase plus Klenow and Exonuclease III significantlyenhanced the blunt ligation. Exonuclease III activity produced bluntends required for ligation of blunt adapters by removing 3′ hydroxyltermini which could be damaged at the 3′ terminus of DNA. ExonucleaseIII also possesses a 3′ phosphatase activity, which makes the 3′terminus accessible to DNA polymerase polishing activity.

Example 3 Temperature Optimization for 5′ Adapter Ligation Using aFAM-Labeled Oligonucleotide Substrate

Rationale: This experiment assessed the temperature dependence and dNTPcomposition on nick translation mediated 5′ adapter ligation.

Materials:

-   -   5′ adapter oligonucleotide for nick-translation (13-144) (Table        1)    -   FAM oligonucleotide substrate (13-581) (Table 1)    -   Oligonucleotide template (13-582) (Table 1)    -   100 mM 2′-deoxynucleoside 5′-triphosphate (dNTP) Set, PCR Grade        (Invitrogen (Life technologies), cat #10297-018)    -   E. coli DNA ligase (New England BioLabs, cat #M0205S)    -   10× E. coli DNA Ligase Reaction Buffer (New England BioLabs)    -   Taq DNA polymerase, concentrated 25 U/ul (Genscript, cat        #E00012)    -   25 bp ladder DNA size marker (Invitrogen (Life technologies),        cat #10488-022)

Method

A first set of nick translation reactions was assembled in a totalvolume of 30 μl, comprising a final concentration of 1× E. coli DNAligase Buffer, 30 pmoles of FAM oligonucleotide substrate, 45 pmoles of5′ adapter oligonucleotide for nick-translation and 45 pmoles ofoligonucleotide template, 200 μM of dTTP or a mix of 200 uM of eachdTTP/dGTP or 200 uM of each dATP/dTTP/dGTP and 2.5 units of Taq DNApolymerase or no Taq DNA polymerase. The reactions were incubated at 30°C., 40° C. or 50° C. for 30 minutes.

A second set of nick translation reactions followed by ligations wereassembled in 30 ul comprising a final concentration of 1× E. coli DNAligase Buffer, 30 pmoles of FAM oligonucleotide substrate, 45 pmoles of5′ adapter oligonucleotide for nick-translation and 45 pmoles ofoligonucleotide template, 200 uM of each dATP/dTTP/dGTP, and 2.5 unitsof Taq DNA polymerase. The reactions were incubated at 50° C., 53° C.,56° C. or 60° C. for 30 minutes. 10 μl of those reactions were taken forgel analysis. 10 units of E. coli ligase were added to the 20 μl leftand incubated at 25° C. for 15 minutes. An additional control reactionwas assembled in 30 μl comprising a final concentration of 1× E. coliDNA ligase Buffer, and 30 pmoles of FAM oligonucleotide substrate. 10 μlof those reactions were mixed with 10 μl of 2× formamide loading buffer(97% formamide, 10 mM EDTA, 0.01% bromophenol blue and 0.01% xylenecyanol), heated at 95° C. for 5 minutes and subsequently run on apre-cast 15% polyacrylamide gel, TBE-Urea (Invitrogen, cat #S11494) inan oven at 65° C., visualized on a Dark reader light box (Clare ChemicalResearch) and photographed using a digital camera.

Results

As shown in FIG. 21, panel A, Taq DNA polymerase elongated the 3′hydroxyl terminus of the 5′ adapter oligonucleotide fornick-translation, removing nucleotides on the FAM oligonucleotidesubstrate by its 5′ flap endonuclease activity. Adding dTTP only (FIG.21, lanes 2, 5, 8, panel A) allowed only the addition of one base at the3′ terminus of the 5′ adapter oligonucleotide for nick-translation,adding dTTP/dGTP (FIG. 21, lanes 3, 6, 9, panel A) allowed the additionof three bases and adding dTTP/dGTP/dATP (FIG. 21, lanes 4, 7, 10, panelA) allowed the addition of four bases which was proportional to thenumber of bases cleaved from the FAM oligonucleotide substrate (FIG. 21,panel A). The number of bases cleaved from the FAM oligonucleotidesubstrate also depended on the temperature in which the reactions takeplace. At 50° C. (FIG. 21, lanes 2 to 4, panel A), the amount of basescleaved from the FAM oligonucleotide substrate was greater than thosecleaved at 40° C. or 30° C. The efficiency of the nick translation andthe amount of FAM oligonucleotide substrate cleaved was also highlydependent on the temperature of the reaction. At 40° C. or 30° C.,adding dTTP only (FIG. 21, lanes 5, 8, panel A), did not allow anycleavage of the FAM oligonucleotide substrate, as observed at 50° C.(FIG. 21, lane 2, panel A). Adding dTTP/dGTP or dTTP/dGTP/dATP allowedsome cleavage at 40° C. (lanes 6 and 7) or 30° C. (lanes 9 and 10) at alower efficacy than at 50° C. (lanes 3 and 4). Lane 1 (FIG. 21, panel A)shows FAM oligonucleotide substrate in the absence of Taq DNApolymerase.

The efficiency of nick translation and the amount of FAM oligonucleotidesubstrate cleaved was highly dependent on the temperature of thereaction. At 60° C., the FAM oligonucleotide substrate was almostentirely processed to smaller species (FIG. 21, lane 4, panel B). TheFAM oligonucleotide substrate cleavage product size also decreased asthe temperature of the reaction increased (FIG. 21, lanes 1 to 4, panelB). Lane 5 (FIG. 21, panel B) shows the FAM oligonucleotide substrate inthe absence of Taq DNA polymerase. During the nick translation reaction,Taq DNA polymerase cleaves the 5′ terminus of the FAM oligonucleotidesubstrate and generates a terminal 5′ phosphate that is essential for E.coli ligase to covalently attach the 3′ terminus of the 5′ adapteroligonucleotide to the 5′ terminus of the FAM oligonucleotide substrate.The ligation efficiency was also dependent on the temperature at whichthe reaction took place. The ligation product was more abundant at 50°C. (lane 6) and almost absent at 60° C. (lane 9), and an intermediateamount of ligation product was generated at 53° C. and 56° C.

Conclusion

During nick translation, the number of bases cleaved from the FAMoligonucleotide substrate depended on the complementary dNTPs introducedin the reaction and the temperature at which the reactions took place.During the nick translation reaction, Taq DNA polymerase cleaves the 5′terminus of the FAM oligonucleotide substrate and generates a terminal5′ phosphate that is essential for E. coli ligase to ligate twofragments. FAM oligonucleotide substrates cleaved by nick translation athigher temperatures were poor substrates for ligation by E. coli ligasebecause of a potential gap formed between the 3′ terminus of the 5′adapter oligonucleotide and the 5′ terminus of the FAM oligonucleotidesubstrate.

Example 4 Analysis of dNTP Composition Effects on 5′ Adapter Ligation

Rationale: This experiment was performed to assess the degree ofnick-translation that occurs in the presence of varied dNTP compositionand the effect on the coupled ligation reaction.

Materials:

-   -   5′ adapter oligonucleotide for nick-translation (13-144) (Table        1)    -   FAM oligonucleotide substrate (13-581) (Table 1)    -   Oligonucleotide template (13-582) (Table 1)    -   100 mM 2′-deoxynucleoside 5′-triphosphate (dNTP) Set, PCR Grade        (Invitrogen (Life technologies), cat #10297-018)    -   25 bp ladder DNA size marker (Invitrogen (Life technologies),        cat #10488-022)    -   E. coli DNA ligase (Enzymatics, cat #L6090L)    -   10× E. coli DNA ligase Buffer (Enzymatics, cat #B6090)    -   Taq-B DNA polymerase (Enzymatics, cat #P7250L)

Method

The reactions were assembled in a total volume of 30 μl, comprising afinal concentration of 1× E. coli DNA ligase Buffer, 30 pmoles of FAMoligonucleotide substrate, 45 pmoles of 5′ adapter oligonucleotide fornick-translation and 45 pmoles of oligonucleotide template, 200 μM ofeach 4 dNTP or a mix of 200 μM of each: dCTP, dTTP, dGTP or dATP, dTTP,dGTP or dATP, dCTP, dGTP or dATP, dTTP, dCTP or no dNTP, 10 units of E.coli ligase and 10 units of Taq-B DNA polymerase. All reactions wereincubated at 40° C. for 30 minutes. 10 μl of those reaction were mixedwith 10 μl of 2× formamide loading buffer (97% formamide, 10 mM EDTA,0.01% bromophenol blue and 0.01% xylene cyanol), heated at 95° C. for 5minutes and subsequently run on a pre-cast 15% polyacrylamide gel,TBE-Urea (Invitrogen, Cat #S11494) in an oven at 65° C., visualized on aDark reader light box (Clare Chemical Research) and photographed using adigital camera (lower panel). Subsequently the gel was stained SYBR®Gold nucleic acid gel stain (Invitrogen, Cat #S11494), visualized on aDark reader light box (Clare Chemical Research) and photographed using adigital camera (upper panel).

Results

The first two lanes of FIG. 22 show control oligonucleotide. In theabsence of Taq-B DNA polymerase, E. coli ligase alone cannot ligate the5′ adapter oligonucleotide to the FAM oligonucleotide substrate becausethe FAM substrate lacks a 5′ phosphate modification (FIG. 22, lane 3).In the presence of Taq-B DNA polymerase and the 4 dNTPs, the 5′ adapteroligonucleotide was extended, forming a new product of 58 bases and theFAM oligonucleotide substrate was displaced and degraded by the 5′ flapendonuclease activity of Taq-B DNA polymerase (FIG. 22, lane 4). In thepresence of E. coli ligase, Taq-B DNA polymerase and dATP/dTTP/dGTP(FIG. 22, lane 7) or dCTP/dTTP/dGTP (FIG. 22, lane 6) or dATP/dTTP/dCTP(FIG. 22, lane 9), nick translation was limited to the addition of four,three or one bases, respectively. With the extension of the 5′ adapter,a flap was formed at the 5′ terminus of the FAM oligonucleotidesubstrate. This flap becomes a substrate for the Taq-B 5′ flapendonuclease activity creating a required 5′ phosphate for ligation. The5′ adapter was ligated to the FAM oligonucleotide substrate forming aproduct of 69 bases. A flap of three or four bases (FIG. 22, lane 6 and7) supported the ligation more efficiently than the one base flap (FIG.22, lane 9). In the presence of E. coli ligase, Taq-B DNA polymerase anddATP/dCTP/dGTP (FIG. 22, lane 8), a faint band corresponding to theligation product was observed. A weak ligation activity may come fromthe incorporation of an “unmatched” base (A C or G instead of T),leading to formation of the flap on some FAM oligonucleotide substrates.In the presence of E. coli ligase, Taq-B DNA polymerase and no dNTP, noligation product was observed. In the presence of E. coli ligase, Taq-BDNA polymerase and the 4 dNTPs, the 5′ adapter was ligated to the FAMoligonucleotide substrate forming a product of 69 bases (FIG. 22, lane5). Since the 5′ adapter and the oligonucleotide template were in excesscompared to the FAM oligonucleotide substrate, a nick translationproduct was also observed at 58 bases (FIG. 22, lane 5, upper panel).However, the same amount of ligation product was observed. The 25 bpladder DNA size marker was loaded on lane M.

Conclusion

Phosphorylation of the 5′ terminus of the FAM oligonucleotide substrateis required for ligation. The polymerase activity of Taq DNA polymerasein the presence of dNTPs is required to perform the extension of the 5′adapter, which creates a flap at the 5′ terminus of the FAMoligonucleotide substrate. This flap is a good substrate for the 5′ flapendonuclease activity of Taq DNA polymerase, generating a perfect 5′phosphate substrate for ligation by E. coli ligase. The ligation occurseven if the flap is only formed by one base. The ligation also occurswhen all four dNTPs are present which does not restrict the length ofthe flap or the extent of nick translation, suggesting that the ligationoccurs immediately after a 5′phosphate is created at the 5′ terminus ofthe FAM oligonucleotide substrate.

Example 5 Coupled Nick Translation-Ligation Reaction with Thermo StableEnzymes

Rationale: This experiment was performed to assess the effect ofreaction temperature and number of units of Taq DNA Polymerase enzyme inthe coupled reaction.

Materials:

-   -   5′ adapter oligonucleotide for nick-translation (13-144) (Table        1)    -   FAM oligonucleotide substrate (13-581) (Table 1)    -   Oligonucleotide template (13-582) (Table 1)    -   100 mM 2′-deoxynucleoside 5′-triphosphate (dNTP) Set, PCR Grade        (Invitrogen (Life technologies), cat #10297-018)    -   Taq DNA ligase (New England BioLabs, cat #M0208S)    -   10× Taq DNA ligase Reaction Buffer (New England BioLabs)    -   Taq DNA polymerase, concentrated 25 U/ul (Genscript, cat        #E00012)

Method

The reactions were assembled in a total volume of 30 μl, comprising afinal concentration of 1× Taq DNA ligase reaction Buffer, 30 pmoles ofFAM oligonucleotide substrate, 45 pmoles of 5′ adapter oligonucleotidefor nick-translation and 45 pmoles of oligonucleotide template, 200 μMof each: dATP, dTTP, dGTP or dTTP, 40 units of Taq DNA ligase, or 80units Taq DNA ligase, or 120 units Taq DNA ligase and 10 units of TaqDNA polymerase. Reactions were incubated at 45° C., 50° C., 55° C., or60° C., for 30 minutes. 10 μl of those reactions were mixed with 10 μlof 2× formamide loading buffer (97% formamide, 10 mM EDTA, 0.01%bromophenol blue and 0.01% xylene cyanol), heated at 95° C. for 5minutes and subsequently run on a pre-cast 15% polyacrylamide gel,TBE-Urea (Invitrogen, Cat #S11494) in an oven at 65° C., visualized on aDark reader light box (Clare Chemical Research) and photographed using adigital camera.

Results

Taq DNA polymerase elongated the 3′ hydroxyl terminus of the 5′ adapteroligonucleotide, removing nucleotides on the FAM oligonucleotidesubstrate by its 5′ flap endonuclease activity. Adding dTTP/dGTP/dATP(FIG. 23, lanes 2 to 5, panel A) or dTTP (FIG. 23, lanes 6 to 9, panelA) allowed the addition of four and one bases, respectively, at the 3′terminus of the 5′ adapter oligonucleotide and the subsequent cleavageof the 5′ terminus of the FAM oligonucleotide substrate. At 60° C. theligation was impaired (FIG. 23, lanes 5 and 9, panel A). The efficiencyof ligation was not affected by adding dTTP/dGTP/dATP (FIG. 23, lanes 2to 5, panel A) or dTTP (FIG. 23, lanes 6 to 9, panel A). The ligationefficiency was dependent on the amount of Taq DNA ligase present in thereaction. The ligation product was more abundant when 120 units of TaqDNA ligase (FIG. 23, lane 4, panel B) were added to the reactioncompared to 40 or 80 units (FIG. 23, lane 2 and 3, panel B,respectively). Lane 1, panel A and lane 1, panel B show controloligonucleotides without enzymes.

Conclusion

During the nick translation reaction, Taq DNA polymerase cleaves the 5′terminus of the FAM oligonucleotide substrate and generates a 5′phosphate terminus essential for Taq DNA ligase between 45° C. and 60°C. to perform ligation. The ligation was reduced at 60° C. Theconcentration of Taq DNA ligase in the reaction also affected theefficiency of the ligation, as more product was observed in the presenceof 120 U enzyme compared to 80 U and 40 U.

Example 6 Coupled Displacement-Cleavage-Ligation Reaction

Rationale: This experiment was performed to demonstrate that eitherthermostable Taq DNA ligase or thermolabile E. coli ligase can becombined with Taq DNA Polymerase in the coupled displacement-cleavageligation reaction.

Materials:

-   -   5′ adapter oligonucleotide for displacement-cleavage (13-156)        (Table 1)    -   FAM oligonucleotide substrate (13-581) (Table 1)    -   Oligonucleotide template (13-582) (Table 1)    -   Taq DNA ligase (New England BioLabs, cat #M0208S)    -   10× Taq DNA ligase Reaction Buffer (New England BioLabs)    -   Taq DNA polymerase, concentrated 25 U/ul (Genscript, cat        #E00012)    -   E. coli DNA ligase (New England BioLabs, cat #M0205S)    -   10× E. coli DNA Ligase Reaction Buffer (New England BioLabs)

Method

The reactions were assembled in a total volume of 30 μl, comprising afinal concentration of 1× E. coli DNA ligase reaction Buffer or 1× TaqDNA ligase reaction Buffer, 30 pmoles of FAM oligonucleotide substrate,45 pmoles of 5′ adapter oligonucleotide for displacement-cleavage and 45pmoles of oligonucleotide template, 10 units of E. coli DNA ligase or 40units Taq DNA ligase, and 10 units of Taq DNA polymerase. Reactions wereincubated at 40° C. or 45° C. for 30 minutes. 10 μl of those reactionswere mixed with 10 μl of 2× formamide loading buffer (97% formamide, 10mM EDTA, 0.01% bromophenol blue and 0.01% xylene cyanol), heated at 95°C. for 5 minutes and subsequently run on a pre-cast 15% polyacrylamidegel, TBE-Urea (Invitrogen, Cat #511494) in an oven at 65° C., visualizedon a Dark reader light box (Clare Chemical Research) and photographedusing a digital camera.

Results

The 5′ adapter oligonucleotide for displacement-cleavage has an extramatching base “T” at is 3′ terminus, which overlaps with the 5′ terminusof the FAM oligonucleotide substrate. When the 3′ terminus of the 5′adapter oligonucleotide displaces the 5′ terminus of the FAMoligonucleotide substrate, the 5′ flap endonuclease activity of Taq DNApolymerase cleaves the 5′ terminus of the FAM oligonucleotide substrateto create a 5′ phosphate which is essential for the ligation with E.coli ligase (FIG. 24, lane 2, panel A) or Taq DNA ligase (FIG. 24, lane2, panel B). Lane 1 for panels A and B show oligonucleotide controlswithout enzymes.

Conclusion

In the absence of dNTPs, no extension of the 5′ adapter occurs. However,Taq DNA polymerase can cleave the 5′ terminus of the FAM oligonucleotidesubstrate and generates a terminal 5′ phosphate that is essential for E.coli DNA ligase or Taq DNA ligase to perform ligation.

Example 7 Coupled Displacement-Cleavage-Ligation Reaction with Either“N” Universal/Degenerate or “T” Substrate-Specific 5′ Adapter 3′Overhang

Rationale: This experiment demonstrates that 5′ adapter ligation using aflap endonuclease can be performed if either the 5′ adapter 3′ terminaloverhang is a sequence-specific match or if it is composed of adegenerate non sequence-specific ‘N’.

Materials:

-   -   5′ adapter oligonucleotide for displacement-cleavage “T”        (13-607) (Table 1)    -   5′ adapter oligonucleotide for displacement-cleavage “N”        (13-596) (Table 1)    -   FAM oligonucleotide substrate (13-581) (Table 1)    -   Oligonucleotide template (13-582) (Table 1)    -   Taq DNA ligase (New England BioLabs, cat #M0208S)    -   10× Taq DNA ligase Reaction Buffer (New England BioLabs)    -   Taq DNA polymerase, concentrated 25 U/ul (Genscript, cat        #E00012)    -   E. coli DNA ligase (New England BioLabs, cat #M0205S)    -   10× E. coli DNA Ligase Reaction Buffer (New England BioLabs)

Method

The reactions were assembled in a total volume of 30 μl, comprising afinal concentration of 1× Taq DNA ligase reaction buffer, 30 pmoles ofFAM oligonucleotide substrate, 45 pmoles of 5′ adapter oligonucleotide“T” or 45 pmoles of 5′ adapter oligonucleotide “N” 1 or 180 pmoles of 5′adapter oligonucleotide “N” or 450 pmoles of 5′ adapter oligonucleotide“N” and 45 pmoles of oligonucleotide template, 40 units Taq DNA ligase,and 10 units of Taq DNA polymerase. Reactions were incubated at 45° C.or 50° C. or 55° C. for 30 minutes or cycling 8 times between 45° C. for3 minutes, 65° C. for 15 seconds. 10 μl of those reactions were mixedwith 10 μl of 2× formamide loading buffer (97% formamide, 10 mM EDTA,0.01% bromophenol blue and 0.01% xylene cyanol), heated at 95° C. for 5minutes and subsequently run on a pre-cast 15% polyacrylamide gel,TBE-Urea (Invitrogen, Cat #S11494) in an oven at 65° C., visualized on aDark reader light box (Clare Chemical Research) and photographed using adigital camera.

Results

When the 5′ adapter oligonucleotide for displacement-cleavage has a “T”at its 3′ terminus matching the oligonucleotide template (FIG. 25, lanes3, 5, 7, panel A), (which overlaps with the 5′ terminus of the FAMoligonucleotide substrate), ligation occurred at a higher rate than whenthe 5′ adapter oligonucleotide had a degenerate “N” base, where duringoligo synthesis, all four nucleotides were present at this position(FIG. 25, lanes 2, 4, 6, panel A), which is only a perfect match to theoligonucleotide template one fourth of the time. Different reactiontemperatures (45° C., 50° C. and 55° C.), were tested without improvingthe ligation using the 5′ adapter oligonucleotide “N” (FIG. 25, lanes 2,4, 6, panel A). Also, different amounts of 5′ adapter oligonucleotide“N” (45 pmoles, 180 pmoles and 450 pmoles), were tested withoutimproving the ligation reaction (FIG. 25, lanes 3 to 5, panel B).However, temperature cycling of the reaction between 45° C. and 65° C.allowed the ligation to occur at the highest rate which was comparableto the “T” matching base 5′ adapter oligonucleotide (FIG. 25, lane 6,panel B). Lane 1 for panels A and B show oligonucleotide controlswithout enzymes.

Conclusion

To allow efficient 5′ adapter ligation coupled to displacement-cleavageusing the 5′ adapter oligonucleotide “N”, cycling between a firsttemperature for Taq DNA ligase to operate and a second temperature wherethe duplex between the oligonucleotide template and the 5′ adapteroligonucleotide “N” could dissociate was critical. The cyclingconditions permitted multiple associations between the 5′ adapteroligonucleotide “N” and the oligonucleotide template where thedisplacement-cleavage reaction occurred only if the 3′ terminal base ofthe 5′ adapter oligonucleotide is a perfect match to the template andcan displace the 5′ terminus of the FAM oligonucleotide substrate.

Example 8 Coupled Nick-Translation-Ligation Reaction Using DNAPolymerase I

Rationale: This experiment demonstrates that a DNA polymerase I, whichpossesses 5′-3′ exonuclease activity, can also participate in the nicktranslation coupled adapter ligation method.

Materials:

-   -   5′ adapter oligonucleotide for nick-translation (13-144) (Table        1)    -   FAM oligonucleotide substrate (13-581) (Table 1)    -   Oligonucleotide template (13-582) (Table 1)    -   100 mM 2′-deoxynucleoside 5′-triphosphate (dNTP) Set, PCR Grade        (Invitrogen (Life technologies), cat #10297-018)    -   25 bp ladder DNA size marker (Invitrogen (Life technologies),        cat #10488-022)    -   E. coli DNA ligase (Enzymatics, cat #L6090L)    -   10× E. coli DNA ligase Buffer (Enzymatics, cat #B6090)    -   Taq-B DNA polymerase (Enzymatics, cat #P7250L)    -   DNA polymerase I (New England Biolabs, cat #M0209S)

Method

The reactions were assembled in a total volume of 30 comprising a finalconcentration of 1× E. coli DNA ligase Buffer, 30 pmoles of FAMoligonucleotide substrate, 45 pmoles of 5′ adapter oligonucleotide fornick-translation and 45 pmoles of oligonucleotide template, 200 μM ofeach 4 dNTPs, 10 units of E. coli ligase and 10 units of Taq-B DNApolymerase or 5 units of DNA polymerase I or 1 unit of DNA polymerase I.Reactions were incubated at 40° C., 18° C., 16° C. or 14° C. for 30minutes. 10 μl of each reaction was mixed with 10 μl of 2× formamideloading buffer (97% formamide, 10 mM EDTA, 0.01% bromophenol blue and0.01% xylene cyanol), heated at 95° C. for 5 minutes and subsequentlyrun on a pre-cast 15% polyacrylamide gel, TBE-Urea (Invitrogen, Cat#S11494) in an oven at 65° C., visualized on a Dark reader light box(Clare Chemical Research) with an without SYBR gold (upper panel andlower panel, respectively), and photographed using a digital camera.

Results

The first lane of FIG. 26 shows the no enzyme control. In the presenceof Taq-B DNA polymerase and E. coli ligase (FIG. 26, lane 2), the 5′adapter oligonucleotide was either ligated to the FAM oligonucleotidesubstrate producing a 69 base product (FIG. 26, lane 2, upper and lowerpanels) or completely extended forming a new product of 58 bases (FIG.26, lane 2, upper panel). The 69 base product was from extension byTaq-B DNA polymerase and formation of a flap at the 5′ end of the FAMoligonucleotide substrate. The Taq-B 5′ flap endonuclease activity cutthe flap and generated a 5′ phosphate that was used by the E. coliligase to complete the ligation. The 58 base product was obtained whenthe FAM oligonucleotide substrate was completely displaced duringextension and degraded by the 5′ flap endonuclease activity of Taq-B DNApolymerase. These two types of products were also formed when Taq-B DNApolymerase was replaced by DNA polymerase I (FIG. 26, lanes 3 to 8)which has a 5′→3′ exonuclease activity that removes nucleotides ahead ofa growing DNA chain one by one and allows nick translation to occur. Thereaction was performed with either 5 units of DNA polymerase I (FIG. 26,lanes 3 to 5) or 1 unit of DNA polymerase I (FIG. 26, lanes 6 to 8). Thereaction with the thermophilic Taq-B DNA polymerase was performed at 40°C. (FIG. 26, lane 2) while the reactions performed with the mesophilicDNA polymerase I were at 18° C. (FIG. 26, lanes 3 and 6), 16° C. (FIG.26, lanes 4 and 7) or 14° C. (FIG. 26, lanes 5 and 8). The 69 baseligation product was obtained in all cases but the addition of only 1unit of DNA polymerase I (FIG. 26, lanes 6 to 8) was more efficient thanwith 5 units (FIG. 26, lanes 3 to 5). This is explained by the verystrong 5′→3′ exonuclease activity of DNA polymerase that causes therapid partial degradation of the FAM oligonucleotide substrate before itcan be ligated. Degradation products were observed in the bottom part ofthe lower panel (FIG. 26, lanes 3 to 5). The 25 bp ladder DNA sizemarker was loaded on lane M.

Conclusion

Taq-B DNA polymerase (thermophilic polymerase) and DNA polymerase I(mesophilic polymerase) can both be used to perform the nick translationmediated ligation but they require different conditions to be fullyactive. They both generated a 69 base product which was the result ofexcision of the 5′ end followed by ligation but they use differentmechanisms. While Taq-B created a flap that was cut to produce therequired 5′ phosphorylated end for the ligation by E. coli ligase, DNApolymerase I removed nucleotides one by one in front of the growingstrand and generated the 5′ phosphorylated nucleotide which was theperfect substrate for E. coli ligase to join the two fragments. DNApolymerase I can be used to perform 5′ adapter ligation mediated by nicktranslation.

Example 9 Polishing is Required for Blunt Ligation of Physically ShearedDNA and Dephosphorylation Prevents the Formation of Chimeric LigationProducts

Rationale: This experiment demonstrates the importance of end polishingand dephosphorylation for blunt ligation of adapters to physicallysheared DNA substrates.

Materials:

-   -   Blue Buffer (Enzymatics, cat #B0110)    -   T4 DNA Ligase (Rapid) (Enzymatics, cat #L6030-HC-L)    -   10× T4 DNA Ligase Buffer (Enzymatics, cat #B6030)    -   100 mM 2′-deoxynucleoside 5′-triphosphate (dNTP) Set, PCR Grade        (Invitrogen (Life technologies), cat #10297-018)    -   Adenosine 5′-Triphosphate (ATP) (New England Biolabs, cat        #P0756S)    -   DNA Polymerase I, Large (Klenow) Fragment (New England Biolabs,        cat #M0210S)    -   T4 DNA polymerase (New England Biolabs, cat #M0203S)    -   T4 Polynucleotide Kinase (New England Biolabs, cat #M0201S)    -   Shrimp alkaline phosphatase (Affymetrix, cat #78390)    -   T4 DNA Ligase (Rapid) (Enzymatics, cat #L6030-HC-L)    -   10× T4 DNA Ligase Buffer (Enzymatics, cat #B6030)    -   E. coli genomic DNA ATCC 11303 strain (Affymetrix, cat #14380)    -   M220 Focused-ultrasonicator, (Covaris, cat #PN 500295)    -   Pippin Prep (Sage Science)    -   DNA Clean & Concentrator-5—(Zymo research, cat #D4004)    -   CDF2010 2% agarose, dye free w/ internal stds (Sage Science)

Method

E. coli gDNA was resuspended in DNA suspension buffer (Teknova, cat#T0227) at a concentration of 100 ng/ul. The DNA was fragmented with theM220 Focused-ultrasonicator to 150 base pairs average size. A tightdistribution of fragmented DNA from ˜150 bp to ˜185 bp was subsequentlysize-selected from a 2% agarose gel using pippin prep.

In a set of reactions A, 100 ng or 500 ng of the size-selected DNA wassubjected to the activity of polishing enzymes. The reactions wereassembled in a total volume of 30 μl, comprising a final concentrationof 1× Blue buffer, 100 uM of each dNTP, 3 units T4 DNA Polymerase, 5units DNA Polymerase I, Large (Klenow) Fragment, 1 mM ATP, 10 units ofT4 Polynucleotide Kinase. The reactions were incubated at 30° C., for 20minutes. The DNA was purified using the DNA Clean & Concentrator-5columns. The DNA was eluted in 15 μl of DNA suspension buffer and asubsequent dephosphorylation reactions B was followed by adapterligation or were placed directly into the ligation reaction withoutdephosphorylation. The dephosphorylation reactions were assembled in a30 μl final volume, including the processed DNA, 1× Blue buffer, and 1unit of shrimp alkaline phosphatase. The reactions were incubated at 37°C., for 10 minutes. The DNA was purified using the DNA Clean &Concentrator-5 columns and eluted in 15 μl of DNA suspension buffer.

In a set of reactions C, 100 ng of the size-selected DNA was subjectedto dephosphorylation followed by polishing or directly to polishing in aset of reaction D. The dephosphorylation reactions were assembled in a30 μl final volume, including the processed DNA, 1× Blue buffer, and 1unit of shrimp alkaline phosphatase. The reactions were incubated at 37°C., for 10 minutes. The DNA was purified using the DNA Clean &Concentrator-5 columns and eluted in 15 μl of DNA suspension buffer. Thepolishing reactions D were assembled in a total volume of 30 μl,comprising a final concentration of 1× Blue buffer, 100 μM of each dNTP,3 units T4 DNA polymerase, 5 units DNA Polymerase I, Large (Klenow)Fragment, (lanes 6 to 7). The DNA was purified using the DNA Clean &Concentrator-5 columns and eluted in 15 μl of DNA suspension buffer.

After purification, all the previous reactions were subject to ligationreactions. Reactions were assembled in a final volume of 30 μl,comprising the processed DNA, 1× T4 DNA ligase reaction buffer and 1200units of T4 DNA ligase. The reactions were incubated at 25° C., for 15minutes. 33 ng of DNA from each ligation was mixed with 2× formamideloading buffer (97% formamide, 10 mM EDTA, 0.01% bromophenol blue and0.01% xylene cyanol), heated at 95° C. for 5 minutes and subsequentlyrun on a pre-cast 15% polyacrylamide gel, TBE-Urea (Invitrogen, Cat#S11494) in an oven at 65° C., stained with SYBR Gold, visualized on aDark reader light box (Clare Chemical Research) and photographed using adigital camera.

Results

Before polishing, physically sheared DNA was not a suitable substratefor ligation to blunt ended adapters by T4 DNA ligase (FIG. 27, lane 1).After polishing with T4 Polynucleotide Kinase, T4 DNA polymerase andKlenow fragment, the DNA ends were blunt, some 5′ termini werephosphorylated and the molecules could concatenate or ligate to eachother as well as to the blunt adapters (FIG. 27, lanes 2 and 4). Thespecies at ˜325 bases, ˜500 bases and over 500 bases correspond to theligation of 2 molecules, 3 molecules and 4 molecules of ˜175 basestogether, respectively (FIG. 27, lanes 2 and 4). The concentration ofDNA influenced the formation of ligation products. At higherconcentration of DNA, the chimeric ligation species of higher molecularweight were more abundant (FIG. 27, lane 4). Treatment of DNA withshrimp alkaline phosphatase after the polishing step impaired concatamerformation between DNA molecules (FIG. 27, lanes 3 and 5). Treatment withshrimp alkaline phosphatase also prevented concatamer formation if itwas performed before the polishing of the fragmented DNA (FIG. 27, lane6). The ligation products observed after polishing with T4 DNApolymerase and Klenow fragment (FIG. 27, lane 7) were not as abundantcompared to the polishing with T4 DNA polymerase, Klenow and T4Polynucleotide Kinase (FIG. 27, lane 2).

Conclusion

Blunt ligation efficiency of physically sheared DNA depended on endpolishing by DNA polymerases. The ligation was also improved by theaddition of T4 Polynucleotide Kinase, which phosphorylated the 5′terminus of the DNA fragments and dephosphorylated the 3′ terminus. Theconcentration of DNA also influenced the amount of ligation and theformation of chimeric products. At higher concentration, DNA is morelikely to form chimeric products in the presences of T4 DNA ligase.Alkaline phosphatases remove 5′ phosphates (which are required forligation) and prevent the formation of chimeric ligation products(concatamers).

Example 10 NGS Libraries have Increased Yield when Prepared Using 5′Base Trimming Coupled to Adapter Ligation Reaction

Rationale: This experiment demonstrates the utility of the reactionspresented in their exemplary application to NGS library construction,particularly the increase in library yield that results from including5′ base trimming coupled to 5′ adapter ligation. Libraries wereconstructed from size-selected sheared DNA so library products could beeasily visualized by gel electrophoresis.

Materials:

-   -   Blue Buffer (Enzymatics, cat #B0110)    -   T4 DNA Ligase (Rapid) (Enzymatics, cat #L6030-HC-L)    -   10× T4 DNA Ligase Buffer (Enzymatics, cat #B6030)    -   100 mM 2′-deoxynucleoside 5′-triphosphate (dNTP) Set, PCR Grade        (Invitrogen (Life technologies), cat #10297-018)    -   Adenosine 5′-Triphosphate (ATP) (New England Biolabs, cat        #P0756S)    -   Klenow Fragment (Enzymatics, cat #P7060L)    -   T4 DNA polymerase (Enzymatics, cat #P7080L)    -   T4 Polynucleotide Kinase (Enzymatics, cat #Y904L)    -   Shrimp alkaline phosphatase (Affymetrix, cat #78390)    -   T4 DNA Ligase (Rapid) (Enzymatics, cat #L6030-HC-L)    -   10× T4 DNA Ligase Buffer (Enzymatics, cat #B6030)    -   3′ Adapler; 1^(st) oligonucleotide 13-501 (Table 1)    -   3′ Adapter; 2^(nd) oligonucleotide 13-712 (Table 1)    -   E. coli genomic DNA ATCC 11303 strain (Affymetrix. cat#14380)    -   M220 Focused-ultrasonicator, (Covaris, cat #PN 500295)    -   E. coli DNA ligase (Enzymatics. cat #L6090L)    -   E. coli DNA ligase buffer (Enzymatics, cat #B6090)    -   Uracil-DNA glycosylase (Enzymatics, cat #G5010L)    -   Taq-B DNA polymerase (Enzymatics, cat #P7250L)    -   5′ adapter oligonucleotide for nick-lranslation (13-489) (Table        1)    -   5′ adapter oligonucleotide for displacement-cleavage (13-595)        (Table 1)    -   1′ aq DNA ligase (Enzymatics, cat #L6060L)    -   SPRIselect (Beckman coulter, cat #B23419)

Methods

E. coli genomic DNA was resuspended in DNA suspension buffer (Teknova,cat #T0227) at a concentration of 100 ng/μl. The DNA was fragmented withthe M220 Focused-ultrasonicator to 150 base pairs average size. A tightdistribution of fragmented DNA from ˜150 bp to ˜185 bp was subsequentlysize-selected on a 2% agarose gel using pippin prep.

100 ng of the size-selected E. coli genomic DNA was used to prepare alibrary with the enhanced adapter ligation method. The polishingreaction was assembled in 30 μl, comprising a final concentration of 1×Blue buffer, 100 μM of each dNTP, 3 units T4 DNA polymerase, 5 units DNAPolymerase I, Large (Klenow) Fragment, 10 units of T4 PolynucleotideKinase. The reaction was incubated at 37° C. for 20 minutes. The DNA waspurified using the DNA Clean & Concentrator-5 and eluted in 15 μl withDNA suspension buffer. The 3′ Adapter ligation reaction was assembled in30 μl including, 1× T4 DNA ligase buffer, 220 pmoles of the 3′ Adapter1st oligonucleotide, 440 pmoles of the 3′ Adapter 2nd oligonucleotide,the 15 μl of DNA purified and 1200 units of T4 DNA ligase. The reactionwas incubated at 25° C. for 15 minutes. The DNA was brought up to a 50μl volume and purified and size selected using 70 μl SPRIselect beads(ratio 1.4×). DNA was eluted in 15 μl of DNA resuspension buffer. Thepartial degradation of the 3′ adapter, annealing of the 5′ adapter,5′-end trimming and ligation of the 5′ adapter all took place in thenext reaction which was assembled in a final volume of 30 μl containing1× E. coli DNA ligase buffer or 1× Taq DNA ligase buffer, 200 μM of eachdNTPs or 200 μM of each dATP, dTTP, dGTP or no dNTPs, 200 pmoles of 5′adapter oligonucleotide for nick-translation or 5′ adapteroligonucleotide for displacement-cleavage, 10 units of E. coli ligase or40 units of Taq DNA ligase, 2 units of uracil-DNA glycosylase, 10 unitsof Taq-B DNA polymerase and 15 μl of the DNA purified after the 3′Adapter ligation reaction. The reaction was incubated at 40° C. or 45°C. for 10 minutes or with 30 cycles of (45° C. for 45 seconds-65° C. for5 seconds) (library 5). The DNA was brought up to a 50 μl volume andpurified and size selected using 40 μl of SPRIselect beads (ratio 0.8×).The DNA was eluted in 20 μl and quantified by qPCR using the KapaLibrary Quantification Kit—Illumina/Universal (cat #KK4824).

Results

The library concentrations were reported on the plot (FIG. 28, panel A)and the libraries were visualized on a 6% polyacrylamide gel byelectrophoresis under denaturing conditions (FIG. 28, panel B). Theinput DNA migrated between ˜150 bases and ˜185 bases (FIG. 28, lane I,panel B). An aliquot was taken after the 3′ adapter ligation step andloaded on the gel. This product migrated between ˜225 to ˜250 bases,which corresponds to the addition of the 64 bases of the 3′ Adapter(FIG. 28, lane L, panel B). The contribution of Taq-B DNA polymerase inremoving one or more bases and exposing a 5′phosphate group at the 5′terminus of the DNA prior to ligation of the 5′ adapter was demonstratedin library 1 vs. 2 (FIG. 28, lanes 1 and 2, panels A and B). Theconcentration of library 1 made without Taq-B (2.6 nM) is three timeslower than library 2 made with Taq-B DNA polymerase (7.9 nM). Even aftertreatment with T4 Polynucleotide Kinase, 75% of the fragmented DNArequired processing of their 5′ termini in order to be ligationcompatible. The finished libraries were also loaded on the gel (FIG. 28,lanes 1 and 2, panel B). These libraries migrated between ˜275 bases and˜300 bases which correspond to the addition of the 58 bases of the 5′adapter oligonucleotide for nick-translation or 5′ adapteroligonucleotide for displacement-cleavage and the 64 bases of the 3′adapter. Library 1 product was present at a lower intensity than thelibrary 2 bands (FIG. 28, panel B). The libraries 3 and 4 were made withdATP, dTTP, dGTP and E. coli ligase or Taq DNA ligase, respectively,during the partial degradation of the 3′ adapter, the annealing of the5′ adapter, the 5′-end trimming and the ligation of the 5′ adapter step.Library 3 concentration (4.8 nM) was about 60% of library 2 (7.9 nM).This loss of 30% in yield is related to the percent of cytosine “C” inthe E. coli genome (25%). Every time the 5′ terminus of the DNAsubstrate is a cytosine, the 5′ adapter oligonucleotide fornick-translation cannot be extended by Taq and the 5′ terminus cannot betrimmed. There is also an extra 6.25% and 1.5% probability to have twoand three consecutive cytosines, respectively, at the 5′ terminus of theDNA substrate. The ligation at 45° C. with Taq DNA ligase (library 4)gave a similar yield (4.8 nM) when compared with E. coli ligase at 40°C. (5.2 nM) (library 3). Library 5, which was made with 5′ adapteroligonucleotide for displacement-cleavage, (4.2 nM) was less efficientthan library 2 made with the 5′ adapter oligonucleotide fornick-translation (7.9 nM).

Conclusion

Libraries were successfully made with the disclosed adapter ligationmethod. The 5′-end DNA trimming by Taq DNA polymerase allows athree-fold increase in the yield of 5′ adapter ligation product whencompared to libraries that have no 5′ end processing step (libraries 1vs 2). Both Taq DNA ligase (library 4) and E. coli ligase (library 3)efficiently ligated the 5′ adapter after the nick-translation. Taq DNAligase also ligated the 5′ adapter after the displacement-cleavage(library 5). Using 4 dNTPs (library 2) instead of 3 (libraries 3 and 4)during the nick-translation may allow the ligation of more DNA substrateto the 5′ adapter.

Example 11 Sequence Analysis of NGS Libraries Prepared Using 5′ BaseTrimming Coupled to Adapter Ligation

Rationale: This experiment demonstrates the utility of the reactionspresented in their exemplary application to NGS library construction.Libraries were constructed from sheared E. coli DNA and then sequencedin order to demonstrate the superior evenness of coverage obtained overa wide base composition of the genome.

Materials:

-   -   Blue Buffer (Enzymatics, cat #B0110)    -   T4 DNA Ligase (Rapid) (Enzymatics, cat #L6030-HC-L)    -   10× T4 DNA Ligase Buffer (Enzymatics, cat #B6030)    -   100 mM 2′-deoxynucleoside 5′-triphosphate (dNTP) Set, PCR Grade        (Invitrogen (Life technologies), cat #10297-018)    -   Adenosine 5′-Triphosphate (ATP) (New England Biolabs, cat        #P0756S)    -   Klenow Fragment (Enzymatics, cat #P7060L)    -   T4 DNA polymerase (Enzymatics, cat #P7080L)    -   T4 Polynucleotide Kinase (Enzymatics, cat #Y904L)    -   Shrimp alkaline phosphatase (Affymetrix, cat #78390)    -   T4 DNA Ligase (Rapid) (Enzymatics, cat #L6030-HC-L)    -   10× T4 DNA Ligase Buffer (Enzymatics, cat #B6030)    -   3′Adapter; 1^(st) oligonucleotide 13-510 (Table 1)    -   3′Adapter; 2^(nd) oligonucleotide 13-712 (Table 1)    -   E. coli genomic DNA ATCC 11303 strain (Affymetrix, cat #14380)    -   M220 Focused-ultrasonicator, (Covaris, cat #PN 500295)    -   E. coli DNA ligase (Enzymatics, cat #L6090L)    -   E. coli DNA ligase buffer (Enzymatics, cat #B6090)    -   Uracil-DNA glycosylase (Enzymatics, cat #G5010L)    -   Taq-B DNA polymerase (Enzymatics, cat #P7250L)    -   5′ adapter oligonucleotide for nick-translation (13-489)    -   SPRIselect (Beckman coulter, cat #B23419)

Method

E. coli genomic DNA was resuspended in DNA suspension buffer (Teknova,cat #T0227) at a concentration of 100 ng/μl. The DNA was fragmented withthe M220 Focused-ultrasonicator to 150 base pairs average size. 100 ngof E. coli covaris genomic DNA was used to prepare a library. A firstreaction of dephosphorylation was assembled in a total volume of 15 μl,comprising a final concentration of 1× Blue buffer, 100 ng of fragmentedE. coli genomic DNA and 1 unit of shrimp alkaline phosphatase. Thereaction was incubated at 37° C. for 10 minutes. The shrimp alkalinephosphatase was inactivated 5 minutes at 65° C. The polishing reactionwas assembled in 30 μl, comprising a final concentration of 1× Bluebuffer, 100 μM of each dNTP, 3 units T4 DNA polymerase, 5 units DNAPolymerase I, Large (Klenow) Fragment and 15 μl of the dephosphorylationreaction. The reaction was incubated at 20° C. for 30 minutes. The DNAwas purified using the DNA Clean & Concentrator-5. The DNA was eluted in15 μl with DNA suspension buffer. The 3′ Adapter ligation reaction wasassembled in 30 μl including, 1× T4 DNA ligase buffer, 220 pmoles of the3′ Adapter 1st oligonucleotide, 440 pmoles of the 3′ Adapter 2ndoligonucleotide, the 15 μl of DNA purified after polishing and 1200units of T4 DNA ligase. The reaction was incubated at 25° C. for 15minutes. After adjusting volume to 50 μl, the DNA was purified and sizedselected using 45 μl SPRIselect beads (ratio 0.9×). DNA was eluted in 15μl of DNA resuspension buffer. The partial degradation of the 3′adapter, annealing of the 5′ adapter, 5′-end DNA trimming and ligationof the 5′ adapter all took place in the next reaction which wasassembled in a final volume of 30 μl containing 1× E. coli DNA ligase,200 μM of each dNTPs, 200 pmoles of 5′ adapter oligonucleotide fornick-translation, 10 units of E. coli ligase, 2 units of uracil-DNAglycosylase, 10 units of Taq-B DNA polymerase and 15 μl of the DNApurified after the 3′ Adapter ligation reaction. The reaction wasincubated at 40° C. for 10 minutes. After adjusting the volume to 50 μl,the DNA was purified using 70 μl of SPRIselect beads (ratio 1.4×). TheDNA was eluted in 20 μl, and quantified by qPCR using the Kapa LibraryQuantification Kit—Illumina/Universal (cat #KK4824). DNA was denatured 5minutes with a final concentration of 0.1 mM of sodium hydroxide and 600μl of 10 pM library was loaded on a MiSeq (Illumina).

Results

The library concentration as quantified by qPCR was 2.8 nM. Pair endreads of 76 bases were generated by the v2 chemistry of the IlluminaMiSeq. 928K/mm² clusters were generated and the Q30 score were 97.8% and96.9% for the first and second read, respectively. The sequence dataquality was assessed using the FastQC report (Babraham Bioinformatics).A summary of the analysis showed 9 green check marks, 2 yellowexclamation points (warning), but no red X (failed) were observed (FIG.29A). The overall % GC of all bases in all sequences was 50%, asexpected for E. coli genome (Green check marks, FIG. 29B). The qualityof the sequence was excellent at every read throughout the 76 basesanalyzed (Green check mark, FIG. 29C). The percentage of each base wasplotted in panel D. The amount of G/C and A/T had <10% difference at anyread (Green check mark, FIG. 29D). The GC content was similar throughoutthe 76 bases analyzed (green check mark, FIG. 29E). The GC content perread across the length of each sequence was compared to a theoreticaldistribution (yellow exclamation point, FIG. 29F). A warning was raisedbecause the sum of the deviations from the normal distribution was foundin more than 15% of the reads (yellow exclamation point, FIG. 29F). Nowarnings were raised for the Per base N content or the Sequence LengthDistribution (summary, FIG. 29A). The sequence duplication level was35.85% (FIG. 29G). A yellow warning was raised because non-uniquesequences make up more than 20% of the total, due to the high level ofcoverage 135× (Yellow exclamation point, FIG. 29G). No overrepresentedsequences or kmer were reported (summary, FIG. 29A). Virtually, noadapter dimer where observed (0.02%, data not shown). The GC bias wasalso evaluated using the Picard CollectGcBiasMetrics as shown in FIG.29H. Evenness of coverage was preserved throughout a broad range of basecomposition. Deviations in coverage were only observed at lower than 10%GC content or higher than 80%. The base quality was over Q25 whichcorrespond to 99.8% accuracy in the base calling. Again, the lowerquality was only observed at extreme low and high GC content.

Conclusion

A library was successfully made using fragmented E. coli genomic DNA.The sequencing demonstrated high quality data and no bias in thecoverage throughout the range of GC content.

Example 12 Oncology Hotspot Panel Combined with Comprehensive Coverageof the TP53 Gene

Rationale: A total of 51 amplicons were designed to cover the entirecoding region of the TP53 gene as well as 30 hotspot loci representingclinically actionable mutations in oncology.

Rationale: This amplicon panel provides proof of concept for thedisclosed method, where the 51 amplicons have significant overlap todemonstrate the absence of the mini-amplicon dominating the reaction, aswell as the evenness of coverage among amplicons that can be achievedusing limited multiplex cycle number. In addition, the high percentageof on target reads demonstrates the specificity of priming becauseprimer dimers and non-specific off target amplification products do notappear in the sequenced library.

Materials:

-   -   Human HapMap genomic DNA (Coriell Institute, NA12878)    -   KAPA HiFi HotStart Uracil+ ReadyMix (KAPA Biosystems, cat        #KK2802)    -   102 Target-specific primers (Table 2)    -   Universal primer containing a 3′ adapter oligonucleotide        truncated sequence and cleavable bases 14-882 (Table 2)    -   E. coli DNA ligase buffer (Enzymatics, cat #B6090)    -   5′ adapter oligonucleotide for adapter ligation step (14-571)    -   5′ part of the 3′ adapter oligonucleotide for adapter ligation        step (14-877)    -   Linker oligonucleotide for adapter ligation step 14-382 (Table        2)    -   E. coli DNA ligase (Enzymatics, cat #L6090L)    -   Uracil-DNA glycosylase (Enzymatics, cat #G5010L)    -   Endonuclease VIII (Enzymatics, cat #Y9080L)    -   Taq-B DNA polymerase (Enzymatics, cat #P7250L)    -   SPRIselect (Beckman coulter, cat #B23419)    -   20% PEG-8000/2.5M NaCl solution for purification steps

Method

Human genomic DNA was diluted in DNA suspension buffer (Teknova, cat#T0227) at a concentration of 2 ng/μl. The DNA was slightly sheared byvortexing for 2 minutes. 10 ng of this sheared genomic DNA was used toprepare a library. A first reaction of amplification was assembled in atotal volume of 30 μl, comprising a final concentration of 1× KAPA HiFiHotStart Uracil+ ReadyMix, 10 ng of sheared human genomic DNA, 300 pmolof the universal primer and a final concentration of 0.85 uM of a mix ofthe 102 target-specific primers present in different ratios. Thefollowing cycling program was run on this reaction: 3 minutes at 95° C.followed by 4 cycles of 20 seconds at 98° C., 5 minutes at 63° C. and 1minute at 72° C. to generate target-specific amplicons and terminated by23 cycles of 20 seconds at 98° C. and 1 minute at 64° C. to producemultiple copies of the target-specific amplicons. After adjusting thevolume to 50 μl, the DNA product was purified using 60 μl of SPRIselectbeads (ratio 1.2×). The beads were resuspended in 50 μl of a 1× reactionmix containing 1× E. coli ligase buffer, 100 pmol of the linkeroligonucleotide, 10 units of E. coli ligase, 10 units of endonucleaseVIII, 2 units of uracil-DNA glycosylase, 20 units of Taq-B DNApolymerase, 100 pmol of the 5′ adapter oligonucleotide and 100 pmol ofthe 5′ part of the 3′ adapter oligonucleotide. The reaction wasincubated at 37° C. for 10 minutes and then purified by adding 42.5 μlof a 20% PEG-8000/2.5M NaCl solution (ratio 0.85×). The DNA was elutedin 20 μl, and quantified by qPCR using the Kapa Library QuantificationKit—Illumina/Universal (cat #KK4824). DNA was denatured 5 minutes with afinal concentration of 0.1 mM of sodium hydroxide and 600 μl of 10 pMlibrary was loaded on a MiSeq (Illumina).

Results

The library concentration as quantified by qPCR was 19.1 nM. Paired endreads of 101 bases were generated by the v2 chemistry of the IlluminaMiSeq. Prior to data analysis, sequence-specific trimming from the 5′end of both read 1 and read 2 is performed to remove synthetic primersequences using the Cutadapt program. The alignment of the paired readsto the human genome and to the targeted regions using BWA-MEM toolshowed exceptional quality data with 98% aligning to targeted regions.Coverage data were also obtained using BEDtools. The coverage uniformitywas 100% meaning that each of the 51 amplicons was represented in thefinal library. The coverage of each individual base in each amplicon wasalso calculated and was higher than 20% of the mean per base coveragemeaning that none of the 51 amplicons were underrepresented in the finalproduct. FIG. 45 depicts the coverage that was obtained for theoverlapping amplicons covering the coding exons of the TP53 gene. FIG.46 depicts a variant call of 18% frequency that was obtained by sequenceanalysis using VarScan and SAMtools.

Conclusion

A targeted amplicon library was successfully made using human genomicDNA. The sequencing demonstrated high quality data.

TABLE 1  SEQ Sequence ID name NO. Sequence 12-900 1AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTAC ACGACGCTCTTCCGATCT 13-426 2AGATCGGAAGAGCGTCGTGTAG/3SpC3/ 13-340 3/5PHOS/AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT/3SpC3/ 13-559 4 ACACGACGCTCTTCCGATCddT 13-5585 ACACGACGCTCTTCCGATCT/3PHOS/ 13-562 6/5PHOS/TGTACCTCACTTCTCATCACTGCT/3FAM/ 13-563 7 AGCAGTGATGAGAAGTGAGGTACA13-561 8 /5PHOS/TGTACCTCACTTCTCATCACTGCT 13-564 9/5FAM/AGCAGTGATGAGAAGTGAGGTACA 13-560 10 TGTACCTCACTTCTCATCACTGCT 13-14411 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT 13-581 12TGTACCTCACTTCTCATCACTGCTGTCATCCGAT/3FAM/ 13-582 13AGCAGTGATGAGAAGTGAGGTACAAGATCGGAAGAGCGT CGTGTAG/3SpC3/ 13-156 14GACTGGAGTTCAGACGTGTGCTCTTCCGATCTT 13-607 15CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGA GTTCAGACGTGTGCTCTTCCGATCTT13-596 16 /5SpC3/C*A*AGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTN 13-501 17/5PHOS/AGATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCT*T*G/3SpC3/ 13-712 18AGACGUGUGCUCUTCCGATCddT 13-489 19/5SpC3/A*A*TGATACGGCGACCACCGAGATCTACACTC TTTCCCTACACGACGCTCTTCCGATCT13-595 20 /5SpC3/A*A*TGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTN 13-510 21/5PHOS/AGATCGGAAGAGCACACGTCTGAACTCCAGTCACGCCAATATCTCGTATGCCGTCTTCTGCT*T*G/3spC3/ *: Phosphorothioated DNA bases/5SpC3/: 5′ C3 spacer (IDT) /3SpC3/: 3′ C3 spacer (IDT) /5PHOS/:5′ Phosphorylation (IDT) /3PHOS/: 3′ Phosphorylation (IDT) /5FAM/:5′ 6-carboxyfluorescein (IDT) /3FAM/: 3′ 6-carboxyfluorescein (IDT) ddT:2′, 3′- Dideoxythymidin (TriLink)

TABLE 2  Oligonucleotides used in Example 12. SEQ Final Sequence IDconcentration name NO Sequence (5′-3′) in PCR (nM) 14-758 22TCAGACGTGTGCTCTTCCGATCTTCTTGCAGCAG  10 nM CCAGA*C*T 14-759 23TCAGACGTGTGCTCTTCCGATCTCCTGCCCTTCC  10 nM AATGGA*T*C 14-760 24TCAGACGTGTGCTCTTCCGATCTCCCCTAGCAGA   5 nM GACCT*G*T 14-864 25TCAGACGTGTGCTCTTCCGATCTGCCCAACCCTT  20 nM GTCCTT*A*C 14-762 26TCAGACGTGTGCTCTTCCGATCTCTGACTGCTCT   5 nM TTTCACCC*A*T 14-763 27TCAGACGTGTGCTCTTCCGATCTGAGCAGCCTCT   5 nM GGCATTC*T*G 14-764 28TCAGACGTGTGCTCTTCCGATCTTGAAGACCCA   5 nM GGTCCAGAT*G*A 14-765 29TCAGACGTGTGCTCTTCCGATCTGCTGCCCTGGT   5 nM AGGTTTTC*T*G 14-766 30TCAGACGTGTGCTCTTCCGATCTCTGGCCCCTGT  15 nM CATCTTC*T*G 14-767 31TCAGACGTGTGCTCTTCCGATCTCAGGCATTGA  15 nM AGTCTCATG*G*A 14-768 32TCAGACGTGTGCTCTTCCGATCTTCCTCCCTGCT  10 nM TCTGTC*T*C 14-769 33TCAGACGTGTGCTCTTCCGATCTCTGTCAGTGGG  10 nM GAACAAGA*A*G 14-885 34TCAGACGTGTGCTCTTCCGATCTGTGCTGTGACT  10 nM GCTTGTA*G*A 14-886 35TCAGACGTGTGCTCTTCCGATCTCTCTGTCTCCT  10 nM TCCTCTTCCT*A*C 14-869 36TCAGACGTGTGCTCTTCCGATCTCTGTGCAGCTG  10 nM TGGGTT*G*A 14-773 37TCAGACGTGTGCTCTTCCGATCTGCTCACCATCG  10 nM CTATCTG*A*G 14-865 38TCAGACGTGTGCTCTTCCGATCTCATGACGGAG   5 nM GTTGTGA*G*G 14-775 39TCAGACGTGTGCTCTTCCGATCTAGCAATCAGT   5 nM GAGGAATCAG*A*G 14-776 40TCAGACGTGTGCTCTTCCGATCTAGCTGGGGCT   5 nM GGAGA*G*A 14-777 41TCAGACGTGTGCTCTTCCGATCTGTCATCCAAAT   5 nM ACTCCACACG*C*A 14-778 42TCAGACGTGTGCTCTTCCGATCTGCATCTTATCC   5 nM GAGTGGAA*G*G 14-779 43TCAGACGTGTGCTCTTCCGATCTCACTGACAACC   5 nM ACCCTTAA*C*C 14-780 44TCAGACGTGTGCTCTTCCGATCTCAGGTAGGAC   5 nM CTGATTTCCTT*A*C 14-781 45TCAGACGTGTGCTCTTCCGATCTTTCTTGCGGAG   5 nM ATTCTCTT*C*C 14-782 46TCAGACGTGTGCTCTTCCGATCTTGGGACGGAA   5 nM CAGCTTTG*A*G 14-783 47TCAGACGTGTGCTCTTCCGATCTCCACCGCTTCT   5 nM TGTCC*T*G 14-784 48TCAGACGTGTGCTCTTCCGATCTGGGTGCAGTTA   5 nM TGCCTC*A*G 14-785 49TCAGACGTGTGCTCTTCCGATCTAGACTTAGTAC   5 nM CTGAAGGGT*G*A 14-786 50TCAGACGTGTGCTCTTCCGATCTTAGCACTGCCC   5 nM AACAACA*C*C 14-787 51TCAGACGTGTGCTCTTCCGATCTCGGCATTTTGA   5 nM GTGTTAGACT*G*G 14-788 52TCAGACGTGTGCTCTTCCGATCTCCTGGTTGTAG  10 nM CTAACTAACT*T*C 14-789 53TCAGACGTGTGCTCTTCCGATCTACCATCGTAAG  10 nM TCAAGTAGCA*T*C 14-790 54TCAGACGTGTGCTCTTCCGATCTATGGTTCTATG   5 nM ACTTTGCCT*G*A 14-791 55TCAGACGTGTGCTCTTCCGATCTAGCAGGCTAG   5 nM GCTAAGCTA*T*G 14-792 56TCAGACGTGTGCTCTTCCGATCTCCTGCTGAAAA  10 nM TGACTGAATATAAACT*T*G 14-793 57TCAGACGTGTGCTCTTCCGATCTGGTCCTGCACC  10 nM AGTAATAT*G*C 14-794 58TCAGACGTGTGCTCTTCCGATCTTGCTTGCTCTG  10 nM ATAGGAAAATG*A*G 14-795 59TCAGACGTGTGCTCTTCCGATCTGGATCCAGAC  10 nM AACTGTTCAAAC*T*G 14-796 60TCAGACGTGTGCTCTTCCGATCTCCAGAAACTG 3.75 nM  CCTCTTGA*C*C 14-797 61TCAGACGTGTGCTCTTCCGATCTGATGTAAGGG 3.75 nM  ACAAGCAG*C*C 14-798 62TCAGACGTGTGCTCTTCCGATCTGAACCAATGG   5 nM ATCGATCTG*C*C 14-799 63TCAGACGTGTGCTCTTCCGATCTGGGGAACTGA   5 nM TGTGACTTA*C*C 14-800 64TCAGACGTGTGCTCTTCCGATCTCTGAGCAAGA   5 nM GGCTTTGG*A*G 14-801 65TCAGACGTGTGCTCTTCCGATCTAACAGTGCAG   5 nM TGTGGAAT*C*C 14-802 66TCAGACGTGTGCTCTTCCGATCTCCACAGAAAC   5 nM CCATGTATGAAG*T*A 14-803 67TCAGACGTGTGCTCTTCCGATCTGTACCCAAAA   5 nM AGGTGACATG*G*A 14-804 68TCAGACGTGTGCTCTTCCGATCTTTTCAGTGTTA  10 nM CTTACCTGTCTTG*T*C 14-805 69TCAGACGTGTGCTCTTCCGATCTGGACTCTGAA  10 nM GATGTACCTATGG*T*C 14-806 70TCAGACGTGTGCTCTTCCGATCTCTCACCATGTC  10 nM CTGACTG*T*G 14-807 71TCAGACGTGTGCTCTTCCGATCTGTGGCACTCTG  10 nM GAAG*C*A 14-808 72TCAGACGTGTGCTCTTCCGATCTGTTACTGAAAG  10 nM CTCAGGGAT*A*G 14-809 73TCAGACGTGTGCTCTTCCGATCTCCACACTTACA  10 nM CATCACTTT*G*C 14-810 74TCAGACGTGTGCTCTTCCGATCTTAGTCTTTCTT  10 nM TGAAGCAGCA*A*G 14-811 75TCAGACGTGTGCTCTTCCGATCTCTAGCTGTGAT  10 nM CCTGAAACTG*A*A 14-812 76TCAGACGTGTGCTCTTCCGATCTTCCTCCTGCAG  20 nM GATTCCT*A*C 14-813 77TCAGACGTGTGCTCTTCCGATCTTGGTGGATGTC  20 nM CTCAAAAG*A*C 14-814 78TCAGACGTGTGCTCTTCCGATCTCAGGATTCTTA  15 nM CAGAAAACAAGTG*G*T 14-815 79TCAGACGTGTGCTCTTCCGATCTTGATGGCAAAT  15 nM ACACAGAGGA*A*G 14-816 80TCAGACGTGTGCTCTTCCGATCTGACGGGTAGA   5 nM GTGTGCG*T*G 14-817 81TCAGACGTGTGCTCTTCCGATCTCGCCACAGAG   5 nM AAGTTGTTG*A*G 14-818 82TCAGACGTGTGCTCTTCCGATCTCGCACTGGCCT  10 nM CATCT*T*G 14-819 83TCAGACGTGTGCTCTTCCGATCTCTTCCAGTGTG  10 nM ATGATGGTG*A*G 14-820 84TCAGACGTGTGCTCTTCCGATCTCATGTGTAACA   5 nM GTTCCTGCA*T*G 14-821 85TCAGACGTGTGCTCTTCCGATCTGGTCAGAGGC   5 nM AAGCAG*A*G 14-822 86TCAGACGTGTGCTCTTCCGATCTTTACTTCTCCC  10 nM CCTCCTC*T*G 14-823 87TCAGACGTGTGCTCTTCCGATCTCTTCCCAGCCT  10 nM GGGCA*T*C 14-824 88TCAGACGTGTGCTCTTCCGATCTGCTGAATGAG   8 nM GCCTTGGA*A*C 14-825 89TCAGACGTGTGCTCTTCCGATCTCTTTCCAACCT   8 nM AGGAAGGC*A*G 14-826 90TCAGACGTGTGCTCTTCCGATCTGCACTGTAATA   5 nM ATCCAGACTGT*G*T 14-827 91TCAGACGTGTGCTCTTCCGATCTCATGTACTGGT   5 nM CCCTCATT*G*C 14-828 92TCAGACGTGTGCTCTTCCGATCTCCTTTCAGGAT  20 nM GGTGGATG*T*G 14-829 93TCAGACGTGTGCTCTTCCGATCTCGACTCCACCA  20 nM GGACT*T*G 14-830 94TCAGACGTGTGCTCTTCCGATCTGTTAACCTTGC   5 nM AGAATGGTCG*A*T 14-831 95TCAGACGTGTGCTCTTCCGATCTCCACGAGAAC   5 nM TTGATCATATTC*A*C 14-832 96TCAGACGTGTGCTCTTCCGATCTCAACAGGTTCT   5 nM TGCTGGTG*T*G 14-833 97TCAGACGTGTGCTCTTCCGATCTATGGTGGGATC   5 nM ATATTCATCTA*C*A 14-836 98TCAGACGTGTGCTCTTCCGATCTAGCTTGTGGAG   5 nM CCTCTTA*C*A 14-837 99TCAGACGTGTGCTCTTCCGATCTGGGACCTTACC   5 nM TTATACACC*G*T 14-838 100TCAGACGTGTGCTCTTCCGATCTCACCATCTCAC   5 nM AATTGCCA*G*T 14-839 101TCAGACGTGTGCTCTTCCGATCTGCTTTCGGAGA   5 nM TGTTGCTTC*T*C 14-840 102TCAGACGTGTGCTCTTCCGATCTGATCCCAGAA   5 nM GGTGAGAAAG*T*T 14-841 103TCAGACGTGTGCTCTTCCGATCTTGAGGTTCAGA   5 nM GCCATG*G*A 14-842 104TCAGACGTGTGCTCTTCCGATCTCTCCAGGAAG  10 nM CCTACGT*G*A 14-843 105TCAGACGTGTGCTCTTCCGATCTGGACATAGTCC  10 nM AGGAGG*C*A 14-844 106TCAGACGTGTGCTCTTCCGATCTCACCGCAGCAT  10 nM GTCAAGA*T*C 14-845 107TCAGACGTGTGCTCTTCCGATCTGACCTAAAGC  10 nM CACCTCCTT*A*C 14-846 108TCAGACGTGTGCTCTTCCGATCTTCCACTATACT  15 nM GACGTCTCCA*A*C 14-847 109TCAGACGTGTGCTCTTCCGATCTACACACGCAA  15 nM AATACTCCTTC*A*G 14-850 110TCAGACGTGTGCTCTTCCGATCTCTGTCCTCACA   5 nM GAGTTCAA*G*C 14-851 111TCAGACGTGTGCTCTTCCGATCTGTTTTTGCAGA   5 nM TGATGGGCT*C*C 14-852 112TCAGACGTGTGCTCTTCCGATCTCTGGACCAAG   5 nM CCCATC*A*C 14-853 113TCAGACGTGTGCTCTTCCGATCTTGTGGCCTTGT   5 nM ACTGCA*G*A 14-854 114TCAGACGTGTGCTCTTCCGATCTCAGTGTGTTCA   5 nM CAGAGACC*T*G 14-855 115TCAGACGTGTGCTCTTCCGATCTGTAGGAAATA   5 nM GCAGCCTCAC*A*T 14-856 116TCAGACGTGTGCTCTTCCGATCTTGTTCCTGATC  15 nM TCCTTAGACA*A*C 14-857 117TCAGACGTGTGCTCTTCCGATCTCTTGCTGCACT  15 nM TCTCACA*C*C 14-858 118TCAGACGTGTGCTCTTCCGATCTTGAAAATTCCA 7.5 nM GTGGCCAT*C*A 14-859 119TCAGACGTGTGCTCTTCCGATCTCAATGAAGAG 7.5 nM AGACCAGA*G*C 14-860 120TCAGACGTGTGCTCTTCCGATCTCCCATACCCTC   5 nM TCAGCGT*A*C 14-861 121TCAGACGTGTGCTCTTCCGATCTGTGGATGTCAG   5 nM GCAGAT*G*C 14-862 122TCAGACGTGTGCTCTTCCGATCTCCCTCCCAGAA  15 nM GGTCTAC*A*T 14-863 123TCAGACGTGTGCTCTTCCGATCTTTTTGACATGG  15 nM TTGGGACTCT*T*G 14-882 124TCAGACGUGUGCUCUUCCGAU*C*U  10 μM 14-382 125 GTGACTGGAGTTCAG —ACGTGT/3PHOS/ 14 877 126 AACTCCAGTCACTAATGCGCATCTCGTATGCCG —TCTTCTGCTTG/3PHOS/ 14-571 127 AATGATACGGCGACCACCGAGATCTACACAGGC —GAAGACACTCTTTCCCTACACGACGCTCTTCCG ATCT *: Phosphorothioated DNA bases(IDT) /3PHOS/: 3′ Phosphorylation (IDT)

The preceding disclosure is supplemented by the following description ofvarious aspects and embodiments of the disclosure, as provided in thefollowing enumerated paragraphs.

A method of producing a processed substrate molecule, the methodcomprising: (i) ligating a first polynucleotide to a 3′ terminus of asubstrate molecule that is at least partially double stranded; (ii)annealing a second polynucleotide to the first polynucleotide underconditions that promote the annealing; (iii) excising at least onenucleotide from the 5′ terminus of the substrate molecule; and then (iv)ligating the second polynucleotide to the 5′ terminus of the doublestranded substrate molecule to produce the processed substrate molecule.

In one embodiment, the method further comprises the step, prior to step(i), of contacting the substrate molecule with a phosphatase enzyme.

In one embodiment, the method further comprises the step of making thesubstrate molecule blunt-ended by contacting the substrate molecule witha polymerase enzyme possessing 3′-5′ exonuclease activity.

In one embodiment, the method further comprises the step of contactingthe substrate molecule with a template-independent polymerase toadenylate the 3′ end of the substrate molecule.

In one embodiment, the substrate molecule is naturally occurring or thesubstrate molecule is synthetic.

In one embodiment, the substrate molecule is naturally occurring.

In one embodiment, the substrate molecule is genomic DNA.

In one embodiment, the genomic DNA is eukaryotic or prokaryotic.

In one embodiment, wherein the genomic DNA is fragmented in vitro or invivo.

In one embodiment, the in vitro fragmenting is performed by a processselected from the group consisting of shearing, cleaving with anendonuclease, sonication, heating, irradiation using an alpha, beta, orgamma source, chemical cleavage in the presence of metal ions, radicalcleavage, and a combination thereof.

In one embodiment, the in vivo fragmenting occurs by a process selectedfrom the group consisting of apoptosis, radiation, and exposure toasbestos.

In one embodiment, the substrate molecule is synthetic and is selectedfrom the group consisting of cDNA, DNA produced by whole genomeamplification, primer extension products comprising at least onedouble-stranded terminus, and a PCR amplicon.

The method of any of the preceding embodiments wherein the firstpolynucleotide is at least partially double stranded and comprisesoligonucleotide 1 and oligonucleotide 2.

In one embodiment, the second polynucleotide anneals to oligonucleotide1.

In one embodiment, the annealing results in a nick, a gap, or anoverlapping base between the second polynucleotide and the substratemolecule.

In one embodiment, the second polynucleotide is contacted with apolymerase, resulting in degradation of oligonucleotide 2.

In one embodiment, oligonucleotide 2 comprises a base that issusceptible to degradation.

In one embodiment, oligonucleotide 2 comprises a blocking group at its3′ end that prevents ligation.

The method of any of the preceding embodiments wherein the secondpolynucleotide comprises a modified base.

In one embodiment, the annealing results in dehybridization ofoligonucleotide 1 and oligonucleotide 2.

The method of any of the preceding embodiments, further comprising: (i)ligating a third polynucleotide to a 3′ terminus of an additionalsubstrate molecule that is at least partially double stranded; (ii)annealing a fourth polynucleotide to the third polynucleotide underconditions that promote the annealing; (iii) excising at least onenucleotide from the 5′ terminus of the additional substrate molecule;and then (iv) ligating the fourth polynucleotide to the 5′ terminus ofthe double stranded additional substrate molecule to produce a processedadditional substrate molecule.

In one embodiment, the first polynucleotide and the third polynucleotideare the same.

In one embodiment, the second polynucleotide and the fourthpolynucleotide are the same.

What is claimed is:
 1. A method of multiplex PCR amplification of aspecific target locus on a nucleic acid substrate for preparing atargeted next generation sequencing library comprising the steps of: (i)combining a plurality of target-specific primers with the nucleic acidsubstrate to yield a single polymerase chain reaction (PCR) reactionmixture, wherein the plurality of target-specific primers comprise afirst forward primer, a second forward primer, a first reverse primerand a second reverse primer, wherein each of the first and secondforward and reverse primers comprise a 3′ complementary sequence that isfully complementary to a sequence of the specific target locus and a 5′noncomplementary sequence that is not complementary to a sequence of thenucleic acid substrate, wherein the 3′ complementary sequence for eachof the first and second forward and reverse primers is different,wherein the 3′ complementary sequence is between 10 and 40 bases inlength, wherein the nucleic acid substrate is human genomic DNA, andwherein the specific target locus is a gene known to have clinicalrelevance in oncology (ii) subjecting the PCR reaction mixture to amultiplex polymerase chain reaction thereby generating at least threeamplicons within the specific target locus, wherein the at least threeamplicons comprise a first amplicon produced by the first forward primerand the first reverse primer, a second amplicon produced by the secondforward primer and the second reverse primer, and a third ampliconproduced by the second forward primer and the first reverse primer,wherein the third amplicon is shorter in length than the first andsecond amplicons, wherein at least a portion of the 5′ noncomplementarysequence of the second forward primer and the first reverse primer isthe same such that each strand of the third amplicon comprises a 3′sequence and a 5′ sequence that are complementary to each other, whereinthe third amplicon possesses overlapping sequence with the first andsecond amplicons, wherein the first amplicon possesses overlappingsequence with the second amplicon, wherein when the third amplicon isdenatured, each strand of the third amplicon forms a secondary structureas a result of the 3′ sequence being complementary to the 5′ sequence,wherein the secondary structure is stable during a primer annealing stepof the multiplex polymerase chain reaction.
 2. The method of claim 1,wherein the multiplex polymerase chain reaction comprises a first seriesof cycle conditions and a second series of cycle conditions, whereineach of the first and second series of cycle conditions include two ormore cycles, wherein the first series of cycle conditions generates thefirst, second, and third amplicons, and wherein the first series ofcycle conditions is different than the second series of cycleconditions.
 3. The method of claim 2, wherein the second series of cycleconditions yield a PCR reaction product, and wherein the second seriesof cycle conditions are sufficient to amplify the first and secondamplicons more efficiently than the third amplicon.
 4. The method ofclaim 3, wherein the first and second amplicons are each present in thePCR reaction product at a greater amount than the third amplicon.
 5. Themethod of claim 2, wherein the first series of cycle conditionscomprises 2 cycles, and wherein the second series of cycle conditionscomprises 14 to more than 20 cycles.
 6. The method of claim 2, whereinthe first series of cycle conditions comprises 4 cycles, and wherein thesecond series of cycle conditions comprises 14 to more than 20 cycles.7. The method of claim 1, wherein the 3′ complementary sequence isbetween 16 and 30 bases in length.
 8. The method of claim 1, wherein thefirst and second amplicon is between 90 bases and 700 bases in length.9. The method of claim 1, wherein the gene known to have clinicalrelevance in oncology is selected from the group consisting of ABU,ABL1, ABL2, ACSL3, AF15Q14, AF1Q, AF3p21, AF5q31, AKAP9, AKT1, AKT2,ALDH2, ALK, ALO17, AMER1, APC, ARHGEF12, ARHH, ARID1A, ARID2, ARNT,ASPSCR1, ASXL1, ATF1, ATIC, ATM, ATP1A1, ATP2B3, ATRX, AXIN1, BAP1,BCL10, BCL11A, BCL11B, BCL2, BCL3, BCLS, BCL6, BCL7A, BCL9, BCOR, BCR,BHD, BIRC3, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD3, BRD4, BRIP1, BTG1,BUB1B, Cl2orf9, Cl5orf21, Cl5orf55, Cl6orf75, C2orf44, CACNA1D, CALR,CAMTA1, CANT1, CARD11, CARS, CASP8, CBFA2T1, CBFA2T3, CBFB, CBL, CBLB,CBLC, CCDC6, CCNBHP1, CCND1, CCND2, CCND3, CCNE1, CD273, CD274, CD74,CD79A, CD79B, CDC73, CDH1, CDH11, CDK12, CDK4, CDK6, CDKN2A, CDKN2C,CDKN2a(p14), CDX2, CEBPA, CEP1, CEP89, CHCHD7, CHEK2, CHIC2, CHN1, CIC,CIITA, CLIP1, CLTC, CLTCL1, CMKOR1, CNOT3, COL1A1, COL2A1, COPEB, COX6C,CREB1, CREB3L1, CREB3L2, CREBBP, CRLF2, CRTC3, CSF3R, CTNNB1, CUX1,CYLD, D1OS170, DAXX, DCTN1, DDB2, DDIT3, DDX10, DDX5, DDX6, DEK, DICER1,DNM2, DNMT3A, DUX4, EBF1, ECT2L, EGFR, EIF3E, EIF4A2, ELF4, ELK4, ELKS,ELL, ELN, EML4, EP300, EPS15, ERBB2, ERC1, ERCC2, ERCC3, ERCC4, ERCC5,ERG, ETV1, ETV4, ETV5, ETV6, EVI1, EWSR1, EXT1, EXT2, EZH2, EZR, FACL6,FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FAS, FBXO11, FBXW7,FCGR2B, FEV, FGFR1, FGFR1OP, FGFR2, FGFR3, FH, FHIT, FIP1L1, FLIT,F1127352, FLT3, FNBP1, FOXA1, FOXL2, FOXO1A, FOXO3A, FOXO4, FOXP1,FSTL3, FUBP1, FUS, FVT1, GAS7, GATA1, GATA2, GATA3, GMPS, GNA1 1, GNAQ,GNAS, GOLGA5, GOPC, GPC3, GPHN, GRAF, H3F3A, H3F3B, HCMOGT-1, HEAB,HERPUD1, HEY1, HIP1, HIS T1H3B , HIS T1H4I, HLA-A, HLF, HLXB 9, HMGA1,HMGA2, HNRNPA2B1, HOOKS, HOXA11, HOXA13, HOXA9, HOXC11, HOXC13, HOXD11,HOXD13, HRAS, HSPCA, HSPCB, IDHL IDH2, IGH\, IGK, IGL, IKZFL IL2, IL21R,IL6ST, IL7R, IRF4, IRTAL ITK, JAKL JAK2, JAK3, JAZFL JUN, KCNJ5, KDM5A,KDM5C, KDM6A, KDR, KIAA1549, KIAA1598, KIF5B, KIT, KLF4, KLK2, KMT2D,KRAS, KTN1, LAF4, LASP1, LCK, LCP1, LCX, LHFP, LIFR, LMNA, LMO1, LMO2,LPP, LRIG3, LSM14A, LYL1, MAF, MAFB, MALAT1, MALT1, MAML2, MAP2K1,MAP2K2, MAP2K4, MAX, MDM2, MDM4, MDS1, MDS2, MECT1, MED12, MEN1, MET,MITF, MKL1, MLF1, MLH1, MLL, MLL3, MLLT1, MLLT10, MLLT2, MLLT3, MLLT4,MLLT6, MLLT7, MN1, MPL, MSF, MSH2, MSH6, MSI2, MSN, MTCP1, MUC1, MUTYH,MYB, MYC, MYCL1, MYCN, MYD88, MYH11, MYH9, MYO5A, MYST4, NAB2, NACA,NBS1, NCOA1, NCOA2, NCOA4, NDRG1, NF1, NF2, NFATC2, NFE2L2, NFIB, NFKB2,NIN, NKX2-1, NONO, NOTCH1, NOTCH2, NPM1, NR4A3, NRAS, NRG1, NSD1, NT5C2,NTRK1, NTRK3, NUMA1, NUP214, NUP98, NUTM2A, NUTM2B, OLIG2, OMD, P2RY8,PAFAH1B2, PALB2, PAX3, PAX5, PAX7, PAX8, PBRM1, PBX1, PCM1, PCSK7,PDE4DIP, PDGFB, PDGFRA, PDGFRB, PERI, PHF6, PHOX2B, PICALM, PIK3CA,PIK3R1, PIM1, PLAG1, PLCG1, PML, PMS1, PMS2, PMX1, PNUTL1, POT1,POU2AF1, POU5F1, PPARG, PPFIBP1, PPP2R1A, PRCC, PRDM1, PRDM16, PRF1,PRKAR1A, PSIP1, PTCH1, PTEN, PTPN11, PTPRB, PTPRC, PTPRK, PWWP2A,RAB5EP, RAC1, RAD21, RAD51L1, RAF1, RALGDS, RANBP17, RAP1GD51, RARA,RB1, RBM15, RECQL4, REL, RET, RNF43, ROS1, RPL10, RPL22, RPL5, RPN1,RSPO2, RSPO3, RUNDC2A, RUNX1, RUNXBP2, SBDS, SDC4, SDH5, SDHB, SDHC,SDHD, 42253, SET, SETBP1, SETD2, SF3B1, SFPQ, SFRS3, SH2B3, SH3GL1, SIL,SLC34A2, SLC45A3, SMAD4, SMARCA4, SMARCB1, SMARCE1, SMO, SOCS1, SOX2,SRGAP3, SRSF2, SS18, SS18L1, SSX1, SSX2, SSX4, STAG2, STATS, STAT5B,STAT6, STK11, STL, SUFU, SUZ12, SYK, TAF15, TALL TAL2, TBL1XR1, TCEA1,TCF1, TCF12, TCF3, TCF7L2, TCL1A, TCL6, TERT, TET2, TFE3, TFEB, TFG,TFPT, TFRC, THRAP3, TIF1, TLX1, TLX3, TMPRSS2, TNFAIP3, TNFRSF14,TNFRSF17, TOP1, TP53, TPM3, TPM4, TPR, TRA, TRAF7, TRB, TRD, TRIM27,TRIM33, TRIP11, TRRAP, TSC1, TSC2, TSHR, TTL, U2AF1, UBRS, USP6, VHL,VTI1A, WAS, WHSC1, WHSC1L1, WIF1, WRN, WT1, WWTR1, XPA, XPC, XPO1,YWHAE, ZCCHC8, ZNF145, ZNF198, ZNF278, ZNF331, ZNF384, ZNF521, ZNF9 andZRSR2.
 10. The method of claim 1, wherein the gene known to haveclinical relevance in oncology is BRCA1 or BRCA2.
 11. The method ofclaim 1, wherein the gene known to have clinical relevance in oncologyis TP53.
 12. The method of claim 1, further comprising (iii) incubatinga sample comprising the first and second amplicons with a 3′ adaptor, a5′ adaptor and a ligase under conditions sufficient to permit ligationof the 3′ adaptor to a 3′ end of the first and second amplicons and 5′adaptor to a 5′ end of the first and second amplicons thereby yielding;the targeted next generation sequencing library.
 13. The method of claim12, further comprising (iv) sequencing the targeted next generationsequencing library.
 14. The method of claim 1, wherein the meltingtemperature of the target-specific primers is from about 60.5° C. toabout 65.5° C.
 15. A method of multiplex PCR amplification of a specifictarget locus on a nucleic acid substrate for preparing a targeted nextgeneration sequencing library comprising the steps of: (i) combining aplurality of target-specific primers with the nucleic acid substrate toyield a single polymerase chain reaction (PCR) reaction mixture, whereinthe plurality of target-specific primers comprise a first forwardprimer, a second forward primer, a first reverse primer and a secondreverse primer, wherein each of the first and second forward and reverseprimers comprise a 3′ complementary sequence that is fully complementaryto a sequence of the specific target locus and a 5′ noncomplementarysequence that is not complementary to a sequence of the nucleic acidsubstrate, wherein the 3′ complementary sequence for each of the firstand second forward and reverse primers is different, wherein the 3′complementary sequence is between 16 and 30 bases in length, and whereinthe specific target locus is a gene known to have clinical relevance inoncology; (ii) subjecting the PCR reaction mixture to a multiplexpolymerase chain reaction thereby generating at least three ampliconswithin the specific target locus, wherein the at least three ampliconscomprise a first amplicon produced by the first forward primer and thefirst reverse primer, a second amplicon produced by the second forwardprimer and the second reverse primer, and a third amplicon produced bythe second forward primer and the first reverse primer, wherein thethird amplicon is shorter in length than the first and second amplicons,wherein at least a portion of the 5′ noncomplementary sequence of thesecond forward primer and the first reverse primer is the same such thateach strand of the third amplicon comprises a 3′ sequence and a 5′sequence that are complementary to each other, wherein the thirdamplicon possesses overlapping sequence with the first and secondamplicons, wherein the first amplicon possesses overlapping sequencewith the second amplicon, wherein when the third amplicon is denatured,each strand of the third amplicon forms a secondary structure as aresult of the 3′ sequence being complementary to the 5′ sequence,wherein at the end of the multiplex polymerase chain reaction, the firstand second amplicons are each present at a greater amount than the thirdamplicon, and wherein the nucleic acid substrate is human genomic DNA.16. The method of claim 15, wherein the gene known to have clinicalrelevance in oncology is selected from the group consisting of ABU,ABL1, ABL2, ACSL3, AF15Q14, AF1Q, AF3p21, AF5q31, AKAP9, AKT1, AKT2,ALDH2, ALK, ALO17, AMER1, APC, ARHGEF12, ARHH, ARID1A, ARID2, ARNT,ASPSCR1, ASXL1, ATF1, ATIC, ATM, ATP1A1, ATP2B3, ATRX, AXIN1, BAP1,BCL10, BCL11A, BCL11B, BCL2, BCL3, BCLS, BCL6, BCL7A, BCL9, BCOR, BCR,BHD, BIRC3, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD3, BRD4, BRIP1, BTG1,BUB1B, Cl2orf9, Cl5orf21, Cl5orf55, Cl6orf75, C2orf44, CACNA1D, CALR,CAMTA1, CANT1, CARD11, CARS, CASP8, CBFA2T1, CBFA2T3, CBFB, CBL, CBLB,CBLC, CCDC6, CCNBHP1, CCND1, CCND2, CCND3, CCNE1, CD273, CD274, CD74,CD79A, CD79B, CDC73, CDH1, CDH11, CDK12, CDK4, CDK6, CDKN2A, CDKN2C,CDKN2a(p14), CDX2, CEBPA, CEP1, CEP89, CHCHD7, CHEK2, CHIC2, CHN1, CIC,CIITA, CLIP1, CLTC, CLTCL1, CMKOR1, CNOT3, COL1A1, COL2A1, COPEB, COX6C,CREB1, CREB3L1, CREB3L2, CREBBP, CRLF2, CRTC3, CSF3R, CTNNB1, CUX1,CYLD, D105170, DAXX, DCTN1, DDB2, DDIT3, DDX10, DDXS, DDX6, DEK, DICER1,DNM2, DNMT3A, DUX4, EBF1, ECT2L, EGFR, EIF3E, EIF4A2, ELF4, ELK4, ELKS,ELL, ELN, EML4, EP300, EPS15, ERBB2, ERC1, ERCC2, ERCC3, ERCC4, ERCCS,ERG, ETV1, ETV4, ETVS, ETV6, EVI1, EWSR1, EXT1, EXT2, EZH2, EZR, FACL6,FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FAS, FBXO11, FBXW7,FCGR2B, FEV, FGFR1, FGFR1OP, FGFR2, FGFR3, FH, FHIT, FIP1L1, FLIT,F1127352, FLT3, FNBP1, FOXA1, FOXL2, FOXO1A, FOXO3A, FOXO4, FOXP1,FSTL3, FUBP1, FUS, FVT1, GAS7, GATA1, GATA2, GATA3, GMPS, GNA1 1, GNAQ,GNAS, GOLGAS, GOPC, GPC3, GPHN, GRAF, H3F3A, H3F3B, HCMOGT-1, HEAB,HERPUD1, HEY1, HIP1, HIS T1H3B , HIS T1H4I, HLA-A, HLF, HLXB 9, HMGA1,HMGA2, HNRNPA2B1, HOOKS, HOXA11, HOXA13, HOXA9, HOXC11, HOXC13, HOXD11,HOXD13, HRAS, HSPCA, HSPCB, IDHL IDH2, IGH\, IGK, IGL, IKZFL IL2, IL21R,IL6ST, IL7R, IRF4, IRTAL ITK, JAKL JAK2, JAK3, JAZFL JUN, KCNJ5, KDM5A,KDM5C, KDM6A, KDR, KIAA1549, KIAA1598, KIF5B, KIT, KLF4, KLK2, KMT2D,KRAS, KTN1, LAF4, LASP1, LCK, LCP1, LCX, LHFP, LIFR, LMNA, LMO1, LMO2,LPP, LRIG3, LSM14A, LYL1, MAF, MAFB, MALAT1, MALT1, MAML2, MAP2K1,MAP2K2, MAP2K4, MAX, MDM2, MDM4, MDS1, MDS2, MECT1, MED12, MEN1, MET,MITF, MKL1, MLF1, MLH1, MLL, MLL3, MLLT1, MLLT10, MLLT2, MLLT3, MLLT4,MLLT6, MLLT7, MN1, MPL, MSF, MSH2, MSH6, MSI2, MSN, MTCP1, MUC1, MUTYH,MYB, MYC, MYCL1, MYCN, MYD88, MYH11, MYH9, MYO5A, MYST4, NAB2, NACA,NBS1, NCOA1, NCOA2, NCOA4, NDRG1, NF1, NF2, NFATC2, NFE2L2, NFIB, NFKB2,NIN, NKX2-1, NONO, NOTCH1, NOTCH2, NPM1, NR4A3, NRAS, NRG1, NSD1, NT5C2,NTRK1, NTRK3, NUMA1, NUP214, NUP98, NUTM2A, NUTM2B, OLIG2, OMD, P2RY8,PAFAH1B2, PALB2, PAX3, PAX5, PAX7, PAX8, PBRM1, PBX1, PCM1, PCSK7,PDE4DIP, PDGFB, PDGFRA, PDGFRB, PERI, PHF6, PHOX2B, PICALM, PIK3CA,PIK3R1, PIM1, PLAG1, PLCG1, PML, PMS1, PMS2, PMX1, PNUTL1, POT1,POU2AF1, POU5F1, PPARG, PPFIBP1, PPP2R1A, PRCC, PRDM1, PRDM16, PRF1,PRKAR1A, PSIP1, PTCH1, PTEN, PTPN11, PTPRB, PTPRC, PTPRK, PWWP2A,RAB5EP, RAC1, RAD21, RAD51L1, RAF1, RALGDS, RANBP17, RAP1GD51, RARA,RB1, RBM15, RECQL4, REL, RET, RNF43, ROS1, RPL10, RPL22, RPL5, RPN1,RSPO2, RSPO3, RUNDC2A, RUNX1, RUNXBP2, SBDS, SDC4, SDH5, SDHB, SDHC,SDHD, 42253, SET, SETBP1, SETD2, SF3B1, SFPQ, SFRS3, SH2B3, SH3GL1, SIL,SLC34A2, SLC45A3, SMAD4, SMARCA4, SMARCB1, SMARCE1, SMO, SOCS1, SOX2,SRGAP3, SRSF2, SS18, SS18L1, SSX1, SSX2, SSX4, STAG2, STATS, STAT5B,STAT6, STK11, STL, SUFU, SUZ12, SYK, TAF15, TALL TAL2, TBL1XR1, TCEA1,TCF1, TCF12, TCF3, TCF7L2, TCL1A, TCL6, TERT, TET2, TFE3, TFEB, TFG,TFPT, TFRC, THRAP3, TIF1, TLX1, TLX3, TMPRSS2, TNFAIP3, TNFRSF14,TNFRSF17, TOP1, TP53, TPM3, TPM4, TPR, TRA, TRAF7, TRB, TRD, TRIM27,TRIM33, TRIP11, TRRAP, TSC1, TSC2, TSHR, TTL, U2AF1, UBRS, USP6, VHL,VTI1A, WAS, WHSC1, WHSC1L1, WIF1, WRN, WT1, WWTR1, XPA, XPC, XPO1,YWHAE, ZCCHC8, ZNF145, ZNF198, ZNF278, ZNF331, ZNF384, ZNF521, ZNF9 andZRSR2.
 17. The method of claim 15, wherein the melting temperature ofthe target-specific primers is from about 60.5° C. to about 65.5° C. 18.The method of claim 15, wherein the gene known to have clinicalrelevance in oncology is BRCA1 or BRCA2.
 19. The method of claim 15,wherein the gene known to have clinical relevance in oncology is TP53.20. A method of multiplex PCR amplification of a specific target locuson a nucleic acid substrate for preparing a targeted next generationsequencing library comprising the steps of: (i) combining a plurality oftarget-specific primers with the nucleic acid substrate to yield asingle polymerase chain reaction (PCR) reaction mixture, wherein theplurality of target-specific primers comprise a first forward primer, asecond forward primer, a first reverse primer and a second reverseprimer, wherein each of the first and second forward and reverse primerscomprise a 3′ complementary sequence that is fully complementary to asequence of the specific target locus and a 5′ noncomplementary sequencethat is not complementary to a sequence of the nucleic acid substrate,wherein the 3′ complementary sequence for each of the first and secondforward and reverse primers is different, wherein the 3′ complementarysequence is between 16 and 30 bases in length; (ii) subjecting the PCRreaction mixture to a multiplex polymerase chain reaction therebygenerating at least three amplicons within the specific target locus,wherein the at least three amplicons comprise a first amplicon producedby the first forward primer and the first reverse primer, a secondamplicon produced by the second forward primer and the second reverseprimer, and a third amplicon produced by the second forward primer andthe first reverse primer, wherein the third amplicon is shorter inlength than the first and second amplicons, wherein at least a portionof the 5′ noncomplementary sequence of the second forward primer and thefirst reverse primer is the same such that each strand of the thirdamplicon comprises a 3′ sequence and a 5′ sequence that arecomplementary to each other, wherein the third amplicon possessesoverlapping sequence with the first and second amplicons, wherein thefirst amplicon possesses overlapping sequence with the second amplicon,wherein when the third amplicon is denatured, each strand of the thirdamplicon forms a secondary structure as a result of the 3′ sequencebeing complementary to the 5′ sequence, wherein the secondary structureis stable during a primer annealing step of the multiplex polymerasechain reaction.